U.S. patent application number 10/633023 was filed with the patent office on 2004-01-29 for nematicidal proteins.
Invention is credited to Foncerrada, Luis, Narva, Kenneth E., Payne, Jewel, Schnepf, H. Ernest, Schwab, George E..
Application Number | 20040018982 10/633023 |
Document ID | / |
Family ID | 27536365 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018982 |
Kind Code |
A1 |
Schnepf, H. Ernest ; et
al. |
January 29, 2004 |
Nematicidal proteins
Abstract
This invention concerns nematicidal proteins obtainable from
Bacillus thuringiensis isolates. The subject invention also
provides various methods of using these proteins for controlling
nematodes.
Inventors: |
Schnepf, H. Ernest; (San
Diego, CA) ; Schwab, George E.; (La Jolla, CA)
; Payne, Jewel; (Davis, CA) ; Narva, Kenneth
E.; (San Diego, CA) ; Foncerrada, Luis;
(Vista, CA) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
2421 N.W. 41ST STREET
SUITE A-1
GAINESVILLE
FL
326066669
|
Family ID: |
27536365 |
Appl. No.: |
10/633023 |
Filed: |
July 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10633023 |
Jul 31, 2003 |
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09738363 |
Dec 15, 2000 |
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6632792 |
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09738363 |
Dec 15, 2000 |
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09076137 |
May 12, 1998 |
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6166195 |
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09076137 |
May 12, 1998 |
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08316301 |
Sep 30, 1994 |
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5753492 |
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08316301 |
Sep 30, 1994 |
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07871510 |
Apr 23, 1992 |
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07871510 |
Apr 23, 1992 |
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07693018 |
May 3, 1991 |
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07871510 |
Apr 23, 1992 |
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07830050 |
Jan 31, 1992 |
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07693018 |
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07565544 |
Aug 10, 1990 |
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07565544 |
Aug 10, 1990 |
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07084653 |
Aug 12, 1987 |
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4948734 |
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10633023 |
Jul 31, 2003 |
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07675772 |
Mar 27, 1991 |
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5262399 |
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07675772 |
Mar 27, 1991 |
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07565544 |
Aug 10, 1990 |
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07675772 |
Mar 27, 1991 |
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07557246 |
Jul 24, 1990 |
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5281530 |
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07557246 |
Jul 24, 1990 |
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07535810 |
Jun 11, 1990 |
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07535810 |
Jun 11, 1990 |
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07084653 |
Aug 12, 1987 |
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4948734 |
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Current U.S.
Class: |
514/4.6 ;
530/324; 530/350 |
Current CPC
Class: |
C07K 14/325 20130101;
A01N 37/46 20130101; A01N 63/50 20200101; C12R 2001/075 20210501;
C12N 1/205 20210501; A01N 63/50 20200101; A01N 63/23 20200101 |
Class at
Publication: |
514/12 ; 530/350;
530/324 |
International
Class: |
A01N 063/00; C07K
014/325 |
Claims
1. An isolated, nematicidal protein wherein said protein comprises
a nematicidal portion of an amino acid sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:10, and SEQ ID NO:12.
2. The protein of claim 1 wherein said protein comprises a
nematicidal portion of SEQ ID No:2.
3. The protein of claim 1 wherein said protein comprises a
nematicidal portion of SEQ ID NO:4.
4. The protein of claim 1 wherein said protein comprises a
nematicidal portion of SEQ ID NO:6.
5. The protein of claim 1 wherein said protein comprises a
nematicidal portion of SEQ ID NO:10.
6. The protein of claim 1 wherein said protein comprises a
nematicidal portion of SEQ ID NO:12.
7. The protein of claim 1 wherein said protein comprises the amino
acid sequence of SEQ ID NO:2.
8. The protein of claim 1 wherein said protein comprises the amino
acid sequence of SEQ ID NO:4.
9. The protein of claim 1 wherein said protein comprises the amino
acid sequence of SEQ ID NO:6.
10. The protein of claim 1 wherein said protein comprises the amino
acid sequence of SEQ ID NO:10.
11. The protein of claim 1 wherein said protein comprises the amino
acid sequence of SEQ ID NO:12.
12. An isolated, nematicidal protein wherein said protein comprises
a nematicidal portion of a .delta.-endotoxin obtainable from a
Bacillus thuringiensis isolate selected from the group consisting
of PS17 (NRRL B-18243), PS33F2 (NRRL B-18244), PS63B (NRRL
B-18246), and PS69D1 (NRRL B-18247).
13. The protein of claim 12 wherein said Bacillus thuringiensis
isolate is PS17 (NRRL B-18243).
14. The protein of claim 12 wherein said Bacillus thuringiensis
isolate is PS33F2 (NRRL B-18244).
15. The protein of claim 12 wherein said Bacillus thuringiensis
isolate is PS63B (NRRL B-18246).
16. The protein of claim 12 wherein said Bacillus thuringiensis
isolate is PS69D1 (NRRL B-18247).
17. The protein of claim 15 wherein said .delta.-endotoxin
comprises the amino acid sequence of SEQ ID NO:20.
18. The protein of claim 15 wherein said .delta.-endotoxin
comprises the amino acid sequence of SEQ ID NO:23.
19. The protein of claim 16 wherein said .delta.-endotoxin
comprises the amino acid sequence of SEQ ID NO:21.
20. The protein of claim 14 wherein said .delta.-endotoxin
comprises the amino acid sequence of SEQ ID NO:22.
21. The protein of claim 12 wherein said protein comprises the
amino acid sequence of a .delta.-endotoxin obtainable from a
Bacillus thuringiensis isolate selected from the group consisting
of PS17 (NRRL B-18243), PS33F2 (NRRL B-18244), PS52A1 (NRRL
B-18245), PS63B (NRRL B-18246), and PS69D1 (NRRL B-18247).
22. The protein of claim 21 wherein said Bacillus thuringiensis
isolate is PS17 (NRRL B-18243).
23. The protein of claim 21 wherein said Bacillus thuringiensis
isolate is PS33F2 (NRRL B-18244).
24. The protein of claim 21 wherein said Bacillus thuringiensis
isolate is PS63B (NRRL B-18246).
25. The protein of claim 21 wherein said Bacillus thuringiensis
isolate is PS69D1 (NRRL B-18247).
26. A method for controlling a nematode pest wherein said method
comprises administering a nematicidal protein to said pest so that
said pest ingests said protein, wherein said protein comprises a
nematicidal portion of an amino acid sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ
ID NO:10.
27. The method of claim 26 wherein said protein comprises a
nematicidal portion of SEQ ID NO:2.
28. The method of claim 26 wherein said protein comprises a
nematicidal portion of SEQ ID NO:4.
29. The method of claim 26 wherein said protein comprises a
nematicidal portion of SEQ ID NO:6.
30. The method of claim 26 wherein said protein comprises a
nematicidal portion of SEQ ID NO:10.
31. The method of claim 26 wherein said protein comprises the amino
acid sequence of SEQ ID NO:2.
32. The method of claim 26 wherein said protein comprises the amino
acid sequence of SEQ ID NO:4.
33. The method of claim 26 wherein said protein comprises the amino
acid sequence of SEQ ID NO:6.
34. The method of claim 26 wherein said protein comprises the amino
acid sequence of SEQ ID NO:10.
35. A method for controlling a nematode pest wherein said method
comprises administering a nematicidal protein to said pest so that
said pest ingests said protein, wherein said protein comprises a
nematicidal portion of a .delta.-endotoxin obtainable from a
Bacillus thuringiensis isolate selected from the group consisting
of PS33F2 (NRRL B-18244), PS52A1 (NRRL B-18245), and PS69D1 (NRRL
B-18247), wherein said .delta.-endotoxin comprises SEQ ID NO:22
when said isolate is PS33F2, said .delta.-endotoxin comprises SEQ
ID NO:19 when said isolate is PS52A1, and said .delta.-endotoxin
comprises SEQ ID NO:21 when said isolate is PS69D1.
36. The method of claim 35 wherein said isolate is PS33F2 and said
.delta.-endotoxin comprises SEQ ID NO:22.
37. The method of claim 35 wherein said isolote is PS69D1 and said
.delta.-endotoxin comprises SEQ ID NO:21.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a division of co-pending application Ser. No.
09/738,363 (filed Dec. 15, 2000) which is a division of application
Ser. No. 09/076,137 (filed on May 12, 1998, which issued as U.S.
Pat. No. 6,166,195 on Dec. 26, 2000) which is a division of
application Ser. No. 08/316,301 (filed on Sep. 30, 1994, which
issued as U.S. Pat. No. 5,753,492 on May 19, 1998) which is a
division of application Ser. No. 07/871,510 (filed on Apr. 23,
1992, now abandoned) which is a continuation-in-part of application
Ser. No. 07/693,018 (filed on May 3, 1991, now abandoned) and a
continuation-in-part of application Ser. No. 07/830,050 (filed on
Jan. 31, 1992, now abandoned). Ser. No. 07/693,018 was a
continuation-in-part of Ser. No. 07/565,544 (filed on Aug. 10,
1990, now abandoned) which is a continuation-in-part of application
Ser. No. 07/084,653 (filed on Aug. 12, 1987, now U.S. Pat. No.
4,948,734). The subject application is also a continuation-in-part
of application Ser. No. 07/675,772 (filed Mar. 27, 1991, now U.S.
Pat. No. 5,262,399) which is a continuation-in-part of Ser. No.
07/565,544 and a continuation-in-part of Ser. No. 07/557,246 (filed
on Jul. 24, 1990, now U.S. Pat. No. 5,281,530). Ser. No. 07/557,246
is a continuation-in-part of Ser. No.07/535,810 (filed Jun. 11,
1990, now abandoned) which is a continuation-in-part of Ser. No.
07/084,653.
BACKGROUND OF THE INVENTION
[0002] Regular use of chemicals to control unwanted organisms can
select for chemical resistant strains. This has occurred in many
species of economically important insects and has also occurred in
nematodes of sheep, goats, and horses. The development of chemical
resistance necessitates a continuing search for new control agents
having different modes of action.
[0003] In recent times, the accepted methodology for control of
nematodes has centered around the drug benzimidazole and its
congeners. The use of these drugs on a wide scale has led to many
instances of resistance among nematode populations (Prichard, R. K.
et al. [1980] "The problem of anthelmintic resistance in
nematodes," Austr. Vet. J. 56:239-251; Coles, G. C. [1986]
"Anthelmintic resistance in sheep," In Veterinary Clinics of North
America: Food Animal Practice, Vol 2:423-432 [Herd, R. P., eds.] W.
B. Saunders, New York). There are more than 100,000 described
species of nematodes.
[0004] The bacterium Bacillus thuringiensis (B.t.) produces a
.delta.-endotoxin polypeptide that has been shown to have activity
against a rapidly growing number of insect species. The earlier
observations of toxicity only against lepidopteran insects have
been expanded with descriptions of B.t. isolates with toxicity to
dipteran and coleopteran insects. These toxins are deposited as
crystalline inclusions within the organism. Many strains of B.t.
produce crystalline inclusions with no demonstrated toxicity to any
insect tested.
[0005] A small number of research articles have been published
about the effects of delta endotoxins from B. thuringiensis species
on the viability of nematode eggs. Bottjer, Bone and Gill
(Experimental Parasitology 60:239-244, 1985) have reported that
B.t. kurstaki and B.t. israelensis were toxic in vitro to eggs of
the nematode Trichostrongylus colubriformis. In addition, 28 other
B.t. strains were tested with widely variable toxicities. The most
potent had LD.sub.50 values in the nanogram range. Ignoffo and
Dropkin (Ignoffo, C. M. and Dropkin, V. H. [1977] J. Kans. Entomol.
Soc. 50:394-398) have reported that the thermostable toxin from
Bacillus thuringiensis (beta exotoxin) was active against a
free-living nematode, Panagrellus redivivus (Goodey); a
plant-parasitic nematode, Meloidogyne incognita (Chitwood); and a
fungus-feeding nematode, Aphelenchus avena (Bastien). Beta exotoxin
is a generalized cytotoxic agent with little or no specificity.
Also, H. Ciordia and W. E. Bizzell (Jour. of Parasitology 47:41
[abstract] 1961) gave a preliminary report on the effects of B.
thuringiensis on some cattle nematodes.
[0006] At the present time there is a need to have more effective
means to control the many nematodes that cause considerable damage
to susceptible hosts. Advantageously, such effective means would
employ biological agents.
BRIEF SUMMMARY OF THE INVENTION
[0007] The subject invention concerns novel toxins active against
nematodes. A further aspect of the invention concerns genes coding
for nematicidal toxins. The subject invention provides the person
skilled in this art with a vast array of nematicidal toxins,
methods for using these toxins, and genes that code for the
toxins.
[0008] One aspect of the invention is the discovery of two
generalized chemical formulae common to a wide range of nematicidal
toxins. These formulae can be used by those skilled in this art to
obtain and identify a wide variety of toxins having the desired
nematicidal activity. The subject invention concerns other
teachings which enable the skilled practitioner to identify and
isolate nematode active toxins and the genes which code therefor.
For example, characteristic features of nematode-active toxin
crystals are disclosed herein. Furthermore, characteristic levels
of amino acid homology can be used to characterize the toxins ofthe
subject invention. Yet another characterizing feature pertains to
immunoreactivity with certain antibodies. Also, nucleotide probes
specific for genes encoding toxins with nematicidal activity are
described.
[0009] In addition to the teachings of the subject invention which
define groups of B.t. toxins with advantageous nematicidal
activity, a further aspect of the subject invention is the
provision of specific nematicidal toxins and the nucleotide
sequences which code for these toxins.
[0010] One aspect of the of the subject invention is the discovery
of two groups of B.t.-derived nematode-active toxins. One group
(CryV) is exemplified by the gene expression products of PS17,
PS33F2 and PS63B, while the other group (CryVI) is exemplified by
the gene expression products of PS52A1 and PS69D1. The organization
of the toxins within each of the two groups can be accomplished by
sequence-specific motifs, overall sequence similarity,
immunoreactivity, and ability to hybridize with specific
probes.
[0011] The genes or gene fragments of the invention encode Bacillus
thuringiensis .delta.-endotoxins which have nematicidal activity.
The genes or gene fragments can be transferred to suitable hosts
via a recombinant DNA vector.
BRIEF DESCRIPTION OF THE SEQUENCES
[0012] SEQ ID NO. 1 discloses the DNA of 17a.
[0013] SEQ ID NO. 2 discloses the amino acid sequence of the toxin
encoded by 17a.
[0014] SEQ ID NO. 3 discloses the DNA of 17b.
[0015] SEQ ID NO. 4 discloses the amino acid sequence of the toxin
encoded by 17b.
[0016] SEQ ID NO. 5 is the nucleotide sequence of a gene from
33F2.
[0017] SEQ ID NO. 6 is the amino acid sequence of the protein
expressed by the gene from 33F2.
[0018] SEQ ID NO. 7 is the nucleotide sequence of a gene from
52A1.
[0019] SEQ ID NO. 8 is the amino acid sequence of the protein
expressed by the gene from 52A1.
[0020] SEQ ID NO. 9 is the nucleotide sequence of a gene from
69D1.
[0021] SEQ ID NO. 10 is the amino acid sequence of the protein
expressed by the gene from 69D1.
[0022] SEQ ID NO. 11 is the nucleotide sequence of a gene from
63B.
[0023] SEQ ID NO. 12 is the amino acid sequence of the protein
expressed by the gene from 63B.
[0024] SEQ ID NO. 13 is the amino acid sequence of a probe which
can be used according to the subject invention.
[0025] SEQ ID NO. 14 is the DNA coding for the amino acid sequence
of SEQ ID NO. 13.
[0026] SEQ ID NO. 15 is the amino acid sequence of a probe which
can be used according to the subject invention.
[0027] SEQ ID NO. 16 is the DNA coding for the amino acid sequence
of SEQ ID NO. 15.
[0028] SEQ ID NO. 17 is the N-terminal amino acid sequence of
17a.
[0029] SEQ ID NO. 18 is the N-terminal amino acid sequence of
17b.
[0030] SEQ ID NO. 19 is the N-terminal amino acid sequence of
52A1.
[0031] SEQ ID NO. 20 is the N-terminal amino acid sequence of
63B.
[0032] SEQ ID NO. 21 is the N-terminal amino acid sequence of
69D1.
[0033] SEQ ID NO. 22 is the N-terminal amino acid sequence of
33F2.
[0034] SEQ ID NO. 23 is an internal amino acid sequence for
63B.
[0035] SEQ ID NO. 24 is a synthetic oligonucleotide derived from
17.
[0036] SEQ ID NO. 25 is an oligonucleotide probe designed from the
N-terminal amino acid sequence of 52A1.
[0037] SEQ ID NO. 26 is the synthetic oligonucleotide probe
designated as 69D1-D.
[0038] SEQ ID NO. 27 is the forward oligonucleotide primer from
63B.
[0039] SEQ ID NO. 28 is the reverse oligonucleotide primer from
63B.
[0040] SEQ ID NO. 29 is the nematode (NEMI) variant of region 5 of
Hofte and Whiteley.
[0041] SEQ ID NO. 30 is the reverse complement primer to SEQ ID NO.
29, used according to the subject invention.
[0042] SEQ ID NO. 31 is a peptide used according to the subject
invention.
[0043] SEQ ID NO. 32 is an oligonucleotide coding for the peptide
of SEQ ID NO. 31.
[0044] SEQ ID NO. 33 is oligonucleotide probe 33F2A.
[0045] SEQ ID NO. 34 is oligonucleotide probe 33F2B.
[0046] SEQ ID NO. 35 is a reverse primer used according to the
subject invention.
[0047] SEQ ID NO. 36 is a forward primer according to the subject
invention.
[0048] SEQ ID NO. 37 is a probe according to the subject
invention.
[0049] SEQ ID NO. 38 is a probe according to the subject
invention.
[0050] SEQ ID NO. 39 is a probe according to the subject
invention.
[0051] SEQ ID NO. 40 is a forward primer according to the subject
invention.
DETAILED DISCLOSURE OF THE INVENTION
[0052] The subject invention concerns a vast array of B.t.
.delta.-endotoxins having nematicidal activity. In addition to
having nematicidal activity, the toxins of the subject invention
will have one or more of the following characteristics:
[0053] 1. An amino acid sequence according to either of the two
generic formulae disclosed herein.
[0054] 2. A high degree of amino acid homology with specific toxins
disclosed herein.
[0055] 3. A DNA sequence encoding the toxin which hybridizes with
probes or genes disclosed herein.
[0056] 4. A nucleotide sequence which can be amplified using
primers disclosed herein.
[0057] 5. A crystal toxin presentation as described herein.
[0058] 6. Immunoreactivity to an antibody raised to a specific
toxin disclosed herein.
[0059] One aspect of the subject invention concerns the discovery
of generic chemical formulae which describe toxins having activity
against nematodes. Two formulae are provided: one which pertains to
nematicidal toxins having molecular weights of between about 45 kDa
and 65 kDa, and the other pertains to larger nematicidal proteins
having molecular weights from about 65 kDa to about 155 kDa. These
formulae represent two different categories of B.t.
.delta.-endotoxins, each of which has activity against nematodes.
The formula describing smaller proteins describes many CryV
proteins, while the formula describing larger proteins describes
many CryVI proteins. A description of these two formulae is as
follows:
[0060] Generic Formula I. This formula describes toxin proteins
having molecular weights from about 65 kDa to about 155 kDa. The
first 650-700 amino acids for proteins in excess of about 75 kDa
and the entire molecule (for proteins of less than about 75 kDa)
have substantially the following sequence:
1 1 MOXXXXXXPX BPYNBLOXXP XZXXXXXXXX OXxXXBXXXE UXBKXBJJXX
XOxxxxZXXZ xXOBXJXBJX XBXXXXBXYX XXVUXZLZLB xxXXXOBPXB 101
ZBXXPBLZBB BXXBXXXXOx xxXUXOXLBX XBOXXBUJBL DJXLXXXXXX XLUXELXXBX
XLXXKXXXXB XExxBXXHXX BXXBXXZXXX KBXXXXBZXX 201 ZBXOXXBXXB
LOEXXXJxxx LXBPXYXBXO XMXLXXXXXX LXXZXOWXXK BxxxxxxxxX XXXXOLXXXK
XXBKXXLXBY XXXXXXBBXX XLXZXZxxZX 301 XXXBXJXXXY XJXMXXX*LE
BXXXXPOBXP EXYxxxZZXL XLXKOKXLBZ XBBXXXXXxx XZBOLXUXXX XOXXXXXXXX
ZXXXBXXXXJ JBXKxUBKBY 401 XXXXXXX*XX *Bx*YXXXBX BUXXXXOXXY
ZXxxxXEPXX ZXXxxxBXXX XPBXXBUXXO XXOXXXXXXX XXOXXXKZXB *XLxxxxxxx
*BXXKX*XXX 501 ZXZXZXZ*XX XLXZXXXXXX XXXXXXXXXX XZXXxxxxx
XLBXXXXPXE XXXXUXLZXX EXXZxUBXXX ZBPBEKxxOZ XXXXBxxBKE WLUZOXXXXL
601 ZPZUZXZBXB OUXOZZXYXB RCRYOZXXXO XBBBUxBXXZ ZXUPLXXUBX
BXXOXEXXOX XXXXUXBXXB KZLXXXXXXB xxxxXxJLPX XXBXBXBOUX 701
ZSSXBXLDKL EBBPBX
[0061] Numbering is for convenience and approximate location
only.
2 Symbols used: A = ala G = gly M = met S = ser C = cys H = his N =
asn T = thr D = asp I = ile P = pro V = val E = glu K = lys Q = gln
W = trp F = phe L = leu R = arg Y = tyr K = K or R E = E or D L = L
or I B = M, L, I, V, or F J = K, R, E, or D O = A or T U = N or Q Z
= G or S X = any naturally occurring amino acid, except C. * = any
naturally occurring amino acid. x = any naturally occurring amino
acid, except C (or complete omission of any amino acids).
[0062] Where a stretch of wild-card amino acids are encountered
(X(n) or x(n) where n>2), repetition of a given amino acid
should be avoided. Similarly, P, C, E, D, K, or R utilization
should be minimized.
[0063] This formula (hereinafter referred to as Generic Formula I)
is exemplified in the current application by the specific toxins
17a, 17b and 63b.
[0064] Generic Formula II. This formula describes toxin proteins
having molecular weights from about 45 kDa to about 65 kDa. Their
primary amino acid structure substantially follows the motif
illustrated below:
3 1 MLBXXXXOBP KHxxxXXXXO XXXXZXKKxx xXZPXXBXXX XXBLLZKXEW
OXBXOYBXOZ XZLPBUJXXB KXHBXLXXJL XLPXJBXULY JBYXXJKXXX 101
XWWUXXLXPLBBKXOUJLXX YZBKXOZJXX KKxxZXXJXB UJJBJULXJU XXJJOXXXKO
XKJBXOKCXL LLKEOJUYJX OOJXBXXXLX XBLXZXUxxx 201 xXJBXZBXXB
UXXLXXBXXX LXXXXZJXZP XXJELLJKBJ XLKXXLEXXL KOEUJLEKKB BXZBXLZPLL
ZBBBYELLEX OOBXXLXXXB JXLXXXLJXO 301 UXJLJKJBKL LZBBUZLXOJ
LJXBXXUZXX OLXBBXKLXZ LWXXLXXULX ULKXOZXXEB XJXXJXJXLX LELXJOXXXW
XXBOXEOXXB XLUZYXXxxx 401 (x)n.sup.a .sup.aWhere n = 0-100
[0065] The symbols used for this formula are the same as those used
for Generic Formula I.
[0066] This formula (hereinafter referred to as Generic Formula II)
is exemplified in the current application by specific toxins 52A1
and 69D1.
[0067] Nematode-active toxins according to the formulae of the
subject invention are specifically exemplified herein by the toxins
encoded by the genes designated 17a, 17b, 63B, 52A1, and 69D1.
Since these toxins are merely exemplary of the toxins represented
by the generic formulae presented herein, it should be readily
apparent that the subject invention further comprises equivalent
toxins (and nucleotide sequences coding for equivalent toxins)
having the same or similar biological activity of the specific
toxins disclosed or claimed herein. These equivalent toxins will
have amino acid homology with the toxins disclosed and claimed
herein. This amino acid homology will typically be greater than
50%, preferably be greater than 75%, and most preferably be greater
than 90%. The amino acid homology will be highest in certain
critical regions of the toxin which account for biological activity
or are involved in the determination of three-dimensional
configuration which ultimately is responsible for the biological
activity. In this regard, certain amino acid substitutions are
acceptable and can be expected if these substitutions are in
regions which are not critical to activity or are conservative
amino acid substitutions which do not affect the three-dimensional
configuration of the molecule. For example, amino acids may be
placed in the following classes: non-polar, uncharged polar, basic,
and acidic. Conservative substitutions whereby an amino acid of one
class is replaced with another amino acid of the same type fall
within the scope of the subject invention so long as the
substitution does not materially alter the biological activity of
the compound. Table 1 provides a listing of examples of amino acids
belonging to each class.
4 TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala,
Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr,
Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His
[0068] In some instances, non-conservative substitutions can also
be made. The critical factor is that these substitutions must not
significantly detract from the biological activity of the toxin.
The information presented in the generic formulae of the subject
invention provides clear guidance to the person skilled in this art
in making various amino acid substitutions.
[0069] Further guidance for characterizing the nematicidal toxins
of the subject invention is provided in Tables 3 and 4, which
demonstrate the relatedness among toxins within each of the
above-noted groups of nematicidal toxins (CryV and CryVI). These
tables show a numeric score for the best matching alignment between
two proteins that reflects: (1) positive scores for exact matches,
(2) positive or negative scores reflecting the likelihood (or not)
of one amino acid substituting for another in a related protein,
and (3) negative scores for the introduction of gaps. A protein
sequence aligned to itself will have the highest possible
score--i.e., all exact matches and no gaps. However, an unrelated
protein or a randomly generated sequence will typically have a low
positive score. Related sequences have scores between the random
background score and the perfect match score.
[0070] The sequence comparisons were made using the algorithm of
Smith and Waterman ([1981] Advances in Applied Mathematics
2:482-489), implemented as the program "Bestfit" in the GCG
Sequence Analysis Software Package Version 7 April 1991. The
sequences were compared with default parameter values (comparison
table: Swgappep.Cmp, Gap weight:3.0, Length weight:0.1) except that
gap limits of 175 residues were applied to each sequence compared.
The program output value compared is referred to as the Quality
score.
[0071] Tables 3 and 4 show the pairwise alignments between the
indicated amino acids of the two classes of nematode-active
proteins CryV and CryVI and representatives of dipteran (CryIV;
Sen, K. et al. [1988] Agric. Biol. Chem. 52:873-878), lepidopteran
and dipteran (CryIIA; Widner and Whiteley [1989] J. Bacteriol.
171:965-974), lepidopteran (CryIA(c); Adang et al. [1981] Gene
36:289-300), and coleopteran (CryIIIA; Herrnstadt et al. [1987]
Gene 57:37-46) proteins.
[0072] Table 2 shows which amino acids were compared from the
proteins of interest.
5 TABLE 2 Protein Amino acids compared 63B 1-692 33F2 1-618 17a
1-677 17b 1-678 CryIV 1-633 CryIIA 1-633 CryIA(c) 1-609 CryIIIA
1-644 69D1 1-395 52A1 1-475
[0073] Table 3 shows the scores prior to adjustment for random
sequence scores.
6 TABLE 3 63B 33F2 17a CryIVA CryIIA CryIA(c) CryIIIA 52A1 69D1 63B
1038 274 338 235 228 232 244 154 122 33F2 927 322 251 232 251 270
157 130 17a 1016 240 240 237 249 152 127 CryIVA 950 245 325 326 158
125 CryIIA 950 244 241 151 132 CryIA(c) 914 367 151 127 CryIIIA 966
150 123 52A1 713 350 69D1 593
[0074] Note that for each nematode-active protein, the highest
score is always with another nematode-active protein. For example,
63B's highest score, aside from itself, is with 17a. Furthermore,
33F2's highest score, aside from itself, is also with 17a.
[0075] Similarly, 52A1 and 69D1 have a higher score versus each
other than with the other proteins.
[0076] Table 4 shows the same analysis after subtraction of the
average score of 50 alignments of random shuffles of the column
sequences with the row sequences.
7 TABLE 4 63B 33F2 17a CryIVA CryIIA CryIA(c) CryIIIA 52A1 69D1 63B
830 81 130 40 32 42 48 0.1 -8.8 33F2 740 128 66 48 72 85 1.4 -2.9
17a 808 45 45 45 54 -0.8 -5.2 CryIVA 759 54 142 138 5.4 -4.1 CryIIA
755 58 53 -2.3 6 CryIA(c) 728 185 3.1 0 CryIIIA 766 -2.3 -6.9 52A1
566 221 69D1 465
[0077] Note that in Table 4 the same relationships hold as in Table
3, i.e., 63B's highest score, aside from itself, is with 17a, and
33F2's highest score, aside from itself, is also with 17a.
[0078] Similarly, 52A1 and 69D1 have a better score versus each
other than with the other proteins.
[0079] Thus, certain toxins according to the subject invention can
be defined as those which have nematode activity and either have an
alignment value (according to the procedures of Table 4) greater
than 100 with 17a or have an alignment value greater than 100 with
52A1. As used herein, the term "alignment value" refers to the
scores obtained above and used to create the scores reported in
Table 4.
[0080] The toxins of the subject invention can also be
characterized in terms of the shape and location of crystal toxin
inclusions. Specifically, nematode-active inclusions typically
remain attached to the spore after cell lysis. These inclusions are
not inside the exosporium, as in previous descriptions of attached
inclusions, but are held within the spore by another mechanism.
Inclusions of the nematode-active isolates are typically amorphic,
generally long and/or multiple. These inclusions are
distinguishable from the larger round/amorphic inclusions that
remain attached to the spore. No B.t. strains that fit this
description have been found to have activity against the
conventional targets--Lepidoptera, Diptera, or Colorado Potato
Beetle. All nematode-active strains fit this description except
one. Thus, there is a very high correlation between this crystal
structure and nematode activity.
[0081] The genes and toxins according to the subject invention
include not only the full length sequences disclosed herein but
also fragments of these sequences, or fusion proteins, which retain
the characteristic nematicidal activity of the sequences
specifically exemplified herein.
[0082] It should be apparent to a person skilled in this art that
genes coding for nematode-active toxins can be identified and
obtained through several means. The specific genes may be obtained
from a culture depository as described below. These genes, or
portions thereof, may be constructed synthetically, for example, by
use of a gene machine. Variations of these genes may be readily
constructed using standard techniques for making point mutations.
Also, fragments of these genes can be made using commercially
available exonucleases or endonucleases according to standard
procedures. For example, enzymes such as Bal31 or site-directed
mutagenesis can be used to systematically cut off nucleotides from
the ends of these genes. Also, genes which code for active
fragments may be obtained using a variety of other restriction
enzymes. Proteases may be used to directly obtain active fragments
of these toxins.
[0083] Equivalent toxins and/or genes encoding these equivalent
toxins can also be located from B.t. isolates and/or DNA libraries
using the teachings provided herein. There are a number of methods
for obtaining the nematode-active toxins of the instant invention
which occur in nature. For example, antibodies to the
nematode-active toxins disclosed and claimed herein can be used to
identify and isolate other toxins from a mixture of proteins.
Specifically, antibodies maybe raised to the portions of the
nematode-active toxins which are most constant and most distinct
from other B.t. toxins. These antibodies can then be used to
specifically identify equivalent toxins with the characteristic
nematicidal activity by immunoprecipitation, enzyme linked
immunoassay (ELISA), or Western blotting. Antibodies to the toxins
disclosed herein, or to equivalent toxins, or fragments of these
toxins, can readily be prepared using standard procedures in this
art. The genes coding for these toxins can then be obtained from
the microorganism.
[0084] A further method for identifying the toxins and genes of the
subject invention is through the use of oligonucleotide probes.
These probes are nucleotide sequences having a detectable label. As
is well known in the art, if the probe molecule and nucleic acid
sample hybridize by forming a strong bond between the two
molecules, it can be reasonably assumed that the probe and sample
are essentially identical. The probe's detectable label provides a
means for determining in a known manner whether hybridization has
occurred. Such a probe analysis provides a rapid method for
identifying nematicidal endotoxin genes of the subject
invention.
[0085] The nucleotide segments which are used as probes according
to the invention can be synthesized by use of DNA synthesizers
using standard procedures. In the use of the nucleotide segments as
probes, the particular probe is labeled with any suitable label
known to those skilled in the art, including radioactive and
non-radioactive labels. Typical radioactive labels include
.sup.32P, .sup.125I, .sup.35S, or the like. A probe labeled with a
radioactive isotope can be constructed from a nucleotide sequence
complementary to the DNA sample by a conventional nick translation
reaction, using a DNase and DNA polymerase. The probe and sample
can then be combined in a hybridization buffer solution and held at
an appropriate temperature until annealing occurs. Thereafter, the
membrane is washed free of extraneous materials, leaving the sample
and bound probe molecules typically detected and quantified by
autoradiography and/or liquid scintillation counting.
[0086] Non-radioactive labels include, for example, ligands such as
biotin or thyroxine, as well as enzymes such as hydrolases or
perioxidases, or the various chemiluminescers such as luciferin, or
fluorescent compounds like fluorescein and its derivatives. The
probe may also be labeled at both ends with different types of
labels for ease of separation, as, for example, by using an
isotopic label at the end mentioned above and a biotin label at the
other end.
[0087] Duplex formation and stability depend on substantial
complementarity between the two strands of a hybrid, and, as noted
above, a certain degree of mismatch can be tolerated. Therefore,
the probes of the subject invention include mutations (both single
and multiple), deletions, insertions of the described sequences,
and combinations thereof, wherein said mutations, insertions and
deletions permit formation of stable hybrids with the target
polynucleotide of interest. Mutations, insertions, and deletions
can be produced in a given polynucleotide sequence in many ways,
and these methods are known to an ordinarily skilled artisan. Other
methods may become known in the future.
[0088] The known methods include, but are not limited to:
[0089] (1) synthesizing chemically or otherwise an artificial
sequence which is a mutation, insertion or deletion of the known
sequence;
[0090] (2) using a probe of the present invention to obtain via
hybridization a new sequence or a mutation, insertion or deletion
of the probe sequence; and
[0091] (3) mutating, inserting or deleting a test sequence in vitro
or in vivo.
[0092] It is important to note that the mutational, insertional,
and deletional variants generated from a given probe may be more or
less efficient than the original probe. Notwithstanding such
differences in efficiency, these variants are within the scope of
the present invention.
[0093] Thus, mutational, insertional, and deletional variants of
the disclosed test sequences can be readily prepared by methods
which are well known to those skilled in the art. These variants
can be used in the same manner as the instant probes so long as the
variants have substantial sequence homology with the probes. As
used herein, substantial sequence homology refers to homology which
is sufficient to enable the variant to function in the same
capacity as the original probe. Preferably, this homology is
greater than 50%; more preferably, this homology is greater than
75%; and most preferably, this homology is greater than 90%. The
degree of homology needed for the variant to function in its
intended capacity will depend upon the intended use of the
sequence. It is well within the skill of a person trained in this
art to make mutational, insertional, and deletional mutations which
are designed to improve the function of the sequence or otherwise
provide a methodological advantage.
[0094] Specific nucleotide probes useful, according to the subject
invention, in the rapid identification of nematode-active genes
are
[0095] (i) DNA coding for a peptide sequence whose single letter
amino acid designation is "REWINGAN" (SEQ ID NO. 13) or variations
thereof which embody point mutations according to the following:
position 1, R or P or K; position 3, W or Y; position 4, I or L;
position 8, N or P; a specific example of such a probe is "AGA(A or
G)T(G or A)(G or T)(A or T)T(A or T)AATGG(A or T)GC(G or T)(A or
C)A(A or T)" (SEQ ID NO. 14);
[0096] (ii) DNA coding for a peptide sequence whose single letter
amino acid designation is "PTFDPDLY" (SEQ ID NO. 15) or variations
thereof which embody point mutations according to the following:
position 3, F or L; position 4, D or Y; position 7, L or H or D; a
specific example of such a probe is "CC(A or T)AC(C or T)TTT(T or
G)ATCCAGAT(C or G)(T or A)TAT" (SEQ ID NO. 16).
[0097] The potential variations in the probes listed is due, in
part, to the redundancy of the genetic code. Because of the
redundancy of the genetic code, i.e., more than one coding
nucleotide triplet (codon) can be used for most of the amino acids
used to make proteins. Therefore different nucleotide sequences can
code for a particular amino acid. Thus, the amino acid sequences of
the B.t. toxins and peptides can be prepared by equivalent
nucleotide sequences encoding the same amino acid sequence of the
protein or peptide. Accordingly, the subject invention includes
such equivalent nucleotide sequences. Also, inverse or complement
sequences are an aspect of the subject invention and can be readily
used by a person skilled in this art. In addition it has been shown
that proteins of identified structure and function may be
constructed by changing the amino acid sequence if such changes do
not alter the protein secondary structure (Kaiser, E. T. and Kezdy,
F. J. [1984] Science 223:249-255). Thus, the subject invention
includes mutants of the amino acid sequence depicted herein which
do not alter the protein secondary structure, or if the structure
is altered, the biological activity is substantially retained.
Further, the invention also includes mutants of organisms hosting
all or part of a toxin encoding a gene of the invention. Such
microbial mutants can be made by techniques well known to persons
skilled in the art. For example, UV irradiation can be used to
prepare mutants of host organisms. Likewise, such mutants may
include asporogenous host cells which also can be prepared by
procedures well known in the art.
[0098] The toxin genes or gene fragments exemplified according to
the subject invention can be obtained from nematode-active B.
thuringiensis (B.t.) isolates designated PS17, PS33F2, PS63B,
PS52A1, and PS69D1. Subcultures of the E. coli host harboring the
toxin genes of the invention were deposited in the permanent
collection of the Northern Research Laboratory, U.S. Department of
Agriculture, Peoria, Ill., USA. The accession numbers are as
follows:
8 Culture Repository No. Deposit Date B.t. isolate PS17 NRRL
B-18243 Jul. 28, 1987 B.t. isolate PS33F2 NRRL B-18244 Jul. 28,
1987 B.t. isolate PS63B NRRL B-18246 Jul. 28, 1987 B.t. isolate
PS52A1 NRRL B-18245 Jul. 28, 1987 B.t. isolate PS69D1 NRRL B-18247
Jul. 28, 1987 E. coli NM522 (pMYC 2316) NRRL B-18785 Mar. 15, 1991
E. coli NM522 (pMYC 2321) NRRL B-18770 Feb. 14, 1991 E. coli NM522
(pMYC 2317) NRRL B-18816 Apr. 24, 1991 E. coli NM522 (pMYC 1627)
NRRL B-18651 May 11, 1990 E. coli NM522 (pMYC 1628) NRRL B-18652
May 11, 1990 E. coli NM522 (pMYC 1642) NRRL B-18961 Apr. 10,
1992
[0099] The subject cultures have been deposited under conditions
that assure that access to the cultures will be available during
the pendency of this patent application to one determined by the
Commissioner of Patents and Trademarks to be entitled thereto under
37 CFR 1.14 and 35 USC 122. The deposits are available as required
by foreign patent laws in countries wherein counterparts of the
subject application, or its progeny, are filed. However, it should
be understood that the availability of a deposit does not
constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
[0100] Further, the subject culture deposits will be stored and
made available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e., they will
be stored with all the care necessary to keep them viable and
uncontaminated for a period of at least five years after the most
recent request for the furnishing of a sample of the deposit, and
in any case, for a period of at least 30 (thirty) years after the
date of deposit or for the enforceable life of any patent which may
issue disclosing the cultures. The depositor acknowledges the duty
to replace the deposits should the depository be unable to furnish
a sample when requested, due to the condition of the deposit(s).
All restrictions on the availability to the public of the subject
culture deposits will be irrevocably removed upon the granting of a
patent disclosing them.
[0101] The novel B.t. genes or gene fragments of the invention
encode toxins which show activity against tested nematodes. The
group of diseases described generally as helminthiasis is due to
infection of an animal host with parasitic worms known as
helminths. Helminthiasis is a prevalent and serious economic
problem in domesticated animals such as swine, sheep, horses,
cattle, goats, dogs, cats and poultry. Among the helminths, the
group of worms described as nematodes causes wide-spread and often
times serious infection in various species of animals. The most
common genera of nematodes infecting the animals referred to above
are Haemonchus, Trichostrongylus, Ostertagia, Nematodirus,
Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,
Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria,
Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria,
Toxascaris, Caenorhabditis and Parascaris. Certain of these, such
as Nematodirus, Cooperia, and Oesophagostomum, attack primarily the
intestinal tract, while others, such as Dictyocaulus are found in
the lungs. Still other parasites may be located in other tissues
and organs of the body.
[0102] The toxins encoded by the novel B.t. genes of the invention
are useful as nematicides for the control of soil nematodes and
plant parasites selected from the genera Bursaphalenchus,
Criconemella, Ditylenchus, Globodera, Helicotylenchus, Heterodera,
Melodoigyne, Pratylenchus, Radolpholus, Rotelynchus, or
Tylenchus.
[0103] Alternatively, because some plant parasitic nematodes are
obligate parasites, genes coding for nematicidal B.t. toxins can be
engineered into plant cells to yield nematode-resistant plants. The
methodology for engineering plant cells is well established (cf.
Nester, E. W., Gordon, M. P., Amasino, R. M. and Yanofsky, M. F.,
Ann. Rev. Plant Physiol. 35:387-399, 1984).
[0104] The B.t. toxins of the invention can be administered orally
in a unit dosage form such as a capsule, bolus or tablet, or as a
liquid drench when used as an anthelmintic in mammals, and in the
soil to control plant nematodes. The drench is normally a solution,
suspension or dispersion of the active ingredient, usually in
water, together with a suspending agent such as bentonite and a
wetting agent or like excipient. Generally, the drenches also
contain an antifoaming agent. Drench formulations generally contain
from about 0.001 to 0.5% by weight of the active compound.
[0105] Preferred drench formulations may contain from 0.01 to 0.1%
by weight, the capsules and boluses comprise the active ingredient
admixed with a carrier vehicle such as starch, talc, magnesium
stearate, or dicalcium phosphate.
[0106] Where it is desired to administer the toxin compounds in a
dry, solid unit dosage form, capsules, boluses or tablets
containing the desired amount of active compound usually are
employed. These dosage forms are prepared by intimately and
uniformly mixing the active ingredient with suitable finely divided
diluents, fillers, disintegrating agents and/or binders such as
starch, lactose, talc, magnesium stearate, vegetable gums and the
like. Such unit dosage formulations may be varied widely with
respect to their total weight and content of the antiparasitic
agent, depending upon the factors such as the type of host animal
to be treated, the severity and type of infection and the weight of
the host.
[0107] When the active compound is to be administered via an animal
feedstuff, it is intimately dispersed in the feed or used as a top
dressing or in the form of pellets which may then be added to the
finished feed or, optionally, fed separately. Alternatively, the
antiparasitic compounds may be administered to animals
parenterally, for example, by intraruminal, intramuscular,
intratracheal, or subcutaneous injection, in which event the active
ingredient is dissolved or dispersed in a liquid carrier vehicle.
For parenteral administration, the active material is suitably
admixed with an acceptable vehicle, preferably of the vegetable oil
variety, such as peanut oil, cotton seed oil and the like. Other
parenteral vehicles, such as organic preparations using solketal,
glycerol, formal and aqueous parenteral formulations, are also
used. The active compound or compounds are dissolved or suspended
in the parenteral formulation for administration; such formulations
generally contain from 0.005 to 5% by weight of the active
compound.
[0108] When the toxins are administered as a component of the feed
of the animals, or dissolved or suspended in the drinking water,
compositions are provided in which the active compound or compounds
are intimately dispersed in an inert carrier or diluent. By inert
carrier is meant one that will not react with the antiparasitic
agent and one that may be administered safely to animals.
Preferably, a carrier for feed administration is one that is, or
may be, an ingredient of the animal ration.
[0109] Suitable compositions include feed premixes or supplements
in which the active ingredient is present in relatively large
amounts and which are suitable for direct feeding to the animal or
for addition to the feed either directly or after an intermediate
dilution or blending step. Typical carriers or diluents suitable
for such compositions include, for example, distillers' dried
grains, corn meal, citrus meal, fermentation residues, ground
oyster shells, wheat shorts, molasses solubles, corn cob meal,
edible bean mill feed, soya grits, crushed limestone and the
like.
[0110] The toxin genes or gene fragments of the subject invention
can be introduced into a wide variety of microbial hosts.
Expression of the toxin gene results, directly or indirectly, in
the intracellular production and maintenance of the nematicide.
With suitable hosts, e.g., Pseudomonas, the microbes can be applied
to the situs of nematodes where they will proliferate and be
ingested by the nematodes. The result is a control of the
nematodes. Alternatively, the microbe hosting the toxin gene can be
treated under conditions that prolong the activity of the toxin
produced in the cell. The treated cell then can be applied to the
environment of target pest(s). The resulting product retains the
toxicity of the B.t. toxin.
[0111] Where the B.t. toxin gene or gene fragment is introduced via
a suitable vector into a microbial host, and said host is applied
to the environment in a living state, it is essential that certain
host microbes be used. Microorganism hosts are selected which are
known to occupy the "phytosphere" (phylloplane, phyllosphere,
rhizosphere, and/or rhizoplane) of one or more crops of interest.
These microorganisms are selected so as to be capable of
successfully competing in the particular environment (crop and
other insect habitats) with the wild-type microorganisms, provide
for stable maintenance and expression of the gene expressing the
polypeptide pesticide, and, desirably, provide for improved
protection of the nematicide from environmental degradation and
inactivation.
[0112] A large number of microorganisms are known to inhabit the
phylloplane (the surface of the plant leaves) and/or the
rhizosphere (the soil surrounding plant roots) of a wide variety of
important crops. These microorganisms include bacteria, algae, and
fungi. Of particular interest are microorganisms, such as bacteria,
e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella,
Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,
Methylophilius, Agrobacterium, Acetobacter, Lactobacillus,
Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi,
particularly yeast, e.g., genera Saccharomyces, Cryptococcus,
Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of
particular interest are such phytosphere bacterial species as
Pseudomonas syringae. Pseudomonas fluorescens, Serratia marcescens,
Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas
spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes
entrophus, and Azotobacter vinlandii; and phytosphere yeast species
such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
[0113] A wide variety of ways are known and available for
introducing the B.t. genes or gene fragments expressing the toxin
into the microorganism host under conditions which allow for stable
maintenance and expression of the gene. The transformants can be
isolated in accordance with conventional ways, usually employing a
selection technique, which allows for selection of the desired
organism as against unmodified organisms or transferring organisms,
when present. The transformants then can be tested for nematicidal
activity.
[0114] Suitable host cells, where the nematicide-containing cells
will be treated to prolong the activity of the toxin in the cell
when the then treated cell is applied to the environment of target
pest(s), may include either prokaryotes or eukaryotes, normally
being limited to those cells which do not produce substances toxic
to higher organisms, such as mammals. However, organisms which
produce substances toxic to higher organisms could be used, where
the toxin is unstable or the level of application sufficiently low
as to avoid any possibility of toxicity to amammalian host. As
hosts, of particular interest will be the prokaryotes and the lower
eukaryotes, such as fungi. Illustrative prokaryotes, both
Gram-negative and -positive, include Enterobacteriaceae, such as
Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae and
Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes
and Ascomycetes, which includes yeast, such as Saccharomyces and
Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,
Aureobasidium, Sporobolomyces, and the like.
[0115] Characteristics of particular interest in selecting a host
cell for purposes of production include ease of introducing the
B.t. gene or gene fragment into the host, availability of
expression systems, efficiency of expression, stability of the
nematicide in the host, and the presence of auxiliary genetic
capabilities. Characteristics of interest for use as a nematicide
microcapsule include protective qualities for the nematicide, such
as thick cell walls, pigmentation, and intracellular packaging or
formation of inclusion bodies; leaf affinity; lack of mammalian
toxicity; attractiveness to pests for ingestion; ease of killing
and fixing without damage to the toxin; and the like. Other
considerations include ease of formulation and handling, economics,
storage stability, and the like.
[0116] Host organisms of particular interest include yeast, such as
Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and
Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp.,
Erwinia sp. and Flavobacterium sp.; or such other organisms as
Escherichia, Lactobacillus sp., Bacillus sp., and the like.
Specific organisms include Pseudomonas aeruginosa, Pseudomonas
fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,
Escherichia coli, Bacillus subtilis, and the like.
[0117] The cell will usually be intact and be substantially in the
proliferative form when treated, rather than in a spore form,
although in some instances spores may be employed.
[0118] Treatment of the microbial cell, e.g., a microbe containing
the B.t. toxin gene or gene fragment, can be by chemical or
physical means, or by a combination of chemical and/or physical
means, so long as the technique does not deleteriously affect the
properties of the toxin, nor diminish the cellular capability in
protecting the toxin. Examples of chemical reagents are
halogenating agents, particularly halogens of atomic no. 17-80.
More particularly, iodine can be used under mild conditions and for
sufficient time to achieve the desired results. Other suitable
techniques include treatment with aldehydes, such as formaldehyde
and glutaraldehyde; anti-infectives, such as zephiran chloride and
cetylpyridinium chloride; alcohols, such as isopropyl and ethanol;
various histologic fixatives, such as Bouin's fixative and Helly's
fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W.
H. Freeman and Company, 1967); or a combination of physical (heat)
and chemical agents that preserve and prolong the activity of the
toxin produced in the cell when the cell is administered to the
host animal. Examples of physical means are short wavelength
radiation such as gamma-radiation and X-radiation, freezing, UV
irradiation, lyophilization, and the like.
[0119] The cells generally will have enhanced structural stability
which will enhance resistance to environmental conditions. Where
the pesticide is in a proform, the method of inactivation should be
selected so as not to inhibit processing of the proform to the
mature form of the pesticide by the target pest pathogen. For
example, formaldehyde will crosslink proteins and could inhibit
processing of the proform of a polypeptide pesticide. The method of
inactivation or killing retains at least a substantial portion of
the bio-availability or bioactivity of the toxin.
[0120] The cellular host containing the B.t. nematicidal gene or
gene fragment maybe grown in any convenient nutrient medium, where
the DNA construct provides a selective advantage, providing for a
selective medium so that substantially all or all of the cells
retain the B.t. gene or gene fragment. These cells may then be
harvested in accordance with conventional ways. Alternatively, the
cells can be treated prior to harvesting.
[0121] The various methods employed in the preparation of the
plasmids and transformation of host organisms are well known in the
art. These procedures are all described in Maniatis, T., Fritsch,
E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York. Thus, it is within
the skill of those in the genetic engineering art to extract DNA
from microbial cells, perform restriction enzyme digestions,
electrophorese DNA fragments, tail and anneal plasmid and insert
DNA, ligate DNA, transform cells, prepare plasmid DNA,
electrophorese proteins, and sequence DNA.
[0122] The B.t. cells may be formulated in a variety of ways. They
may be employed as wettable powders, granules or dusts, by mixing
with various inert materials, such as inorganic minerals
(phyllosilicates, carbonates, sulfates, phosphates, and the like)
or botanical materials (powdered corncobs, rice hulls, walnut
shells, and the like). The formulations may include
spreader-sticker adjuvants, stabilizing agents, other pesticidal
additives, or surfactants. Liquid formulations may be aqueous-based
or non-aqueous and employed as foams, gels, suspensions,
emulsifiable concentrates, or the like. The ingredients may include
rheological agents, surfactants, emulsifiers, dispersants, or
polymers.
[0123] The nematicide concentration will vary widely depending upon
the nature of the particular formulation, particularly whether it
is a concentrate or to be used directly. The nematicide will be
present in at least 1% by weight and may be 100% by weight. The dry
formulations will have from about 1-95% by weight of the nematicide
while the liquid formulations will generally be from about 1-60% by
weight of the solids in the liquid phase. The formulations will
generally have from about 10.sup.2 to about 10.sup.4 cells/mg.
These formulations will be administered at about 50 mg (liquid or
dry) to 1 kg or more per hectare.
[0124] The formulations can be applied to the environment of the
nematodes, e.g., plants, soil or water, by spraying, dusting,
sprinkling, or the like.
[0125] Following are examples which illustrate procedures,
including the best mode, for practicing the invention. These
examples should not be construed as limiting. All percentages are
by weight and all solvent mixture proportions are by volume unless
otherwise noted.
EXAMPLE 1
[0126] Culturing B.t. Isolates of the Invention
[0127] A subculture of a B.t. isolate can be used to inoculate the
following medium, a peptone, glucose, salts medium.
9 Bacto Peptone 7.5 g/l Glucose 1.0 g/l KH.sub.2PO.sub.4 3.4 g/l
K.sub.2HPO.sub.4 4.35 g/l Salts Solution 5.0 ml/l CaCl.sub.2
Solution 5.0 ml/l Salts Solution (100 ml) MgSO.sub.4.7H.sub.2O 2.46
g MnSO.sub.4.H.sub.2O 0.04 g ZnSO.sub.4.7H.sub.2O 0.28 g
FeSO.sub.4.7H.sub.2O 0.40 g CaCl.sub.2 Solution (100 ml)
CaCl.sub.2.2H.sub.2O 3.66 g pH 7.2
[0128] The salts solution and CaCl.sub.2 solution are
filter-sterilized and added to the autoclaved and cooked broth at
the time of inoculation. Flasks are incubated at 30.degree. C. on a
rotary shaker at 200 rpm for 64 hr.
EXAMPLE 2
[0129] Purification of Protein and Amino Acid Sequencing
[0130] The B.t. isolates PS17, PS63B, PS52A1, and PS69D1 were
cultured as described in Example 1. The parasporal inclusion bodies
were partially purified by sodium bromide (28-38%) isopycnic
gradient centrifugation (Pfannenstiel, M. A., E. J. Ross, V. C.
Kramer, and K. W. Nickerson [1984] FEMS Microbiol. Lett. 21:39).
The proteins toxic for the nematode Caenorhabditis elegans were
bound to PVDF membranes (Millipore, Bedford, Mass.) by western
blotting techniques (Towbin, H., T. Staehlelin, and K. Gordon
[1979] Proc. Natl. Acad. Sci. USA 76:4350) and the N-terminal amino
acid sequences were determined by the standard Edman reaction with
an automated gas-phase sequenator (Hunkapiller, M. W., R. M.
Hewick, W. L. Dreyer, and L. E. Hood [1983] Meth. Enzymol. 91:399).
The sequences obtained were:
10 PS17a: A I L N E L Y P S V P Y N V (SEQ ID NO.17) PS17b: A I L N
E L Y P S V P Y N V (SEQ ID NO.18) PS52A1: M I I D S K T T L P R H
S L I N T (SEQ ID NO.19) PS63B: Q L Q A Q P L I P Y N V L A (SEQ ID
NO.20) PS69D1: M I L G N G K T L P K H I R L A H I F A T Q N S (SEQ
ID NO.21) PS33F2: A T L N E V Y P V N (SEQ ID NO.22)
[0131] In addition, internal amino acid sequence data were derived
for PS63B. The toxin protein was partially digested with
Staphylococcus aureus V8 protease (Sigma Chem. Co., St. Louis, Mo.)
essentially as described (Cleveland, D. W., S. G. Fischer, M. W.
Kirschner, and U. K. Laemmli [1977] J. Biol. Chem. 252:1102). The
digested material was blotted onto PVDF membrane and a ca. 28 kDa
limit peptide was selected for N-terminal sequencing as described
above. The sequence obtained was:
[0132] PS63B(2) V Q R I L D E K L S F Q L I K (SEQ ID NO. 23)
[0133] From these sequence data oligonucleotide probes were
designed by utilizing a codon frequency table assembled from
available sequence data of other B.t. toxin genes. The probes were
synthesized on an Applied Biosystems, Inc. DNA synthesis
machine.
[0134] Protein purification and subsequent amino acid analysis of
the N-terminal peptides listed above has led to the deduction of
several oligonucleotide probes for the isolation of toxin genes
from nematicidal B.t. isolates. RFLP analysis of restricted total
cellular DNA using radiolabeled oligonucleotide probes has
elucidated different genes or gene fragments.
EXAMPLE 3
[0135] Cloning of Novel Toxin Genes and Transformation into
Escherichia coli
[0136] Total cellular DNA was prepared by growing the cells B.t.
PS17 to a low optical density (OD.sub.600=1.0) and recovering the
cells by centrifugation. The cells were protoplasted in TES buffer
(30 mM Tris-Cl, 10 mM EDTA, 50 mM NaCl, pH=8.0) containing 20%
sucrose and 50 mg/ml lysozyme. The protoplasts were lysed by
addition of SDS to a final concentration of 4%. The cellular
material was precipitated overnight at 4.degree. C. in 100 mM
(final concentration) neutral potassium chloride. The supernate was
extracted twice with phenol/chloroform (1:1). The DNA was
precipitated with ethanol and purified by isopycnic banding on a
cesium chloride-ethidium bromide gradient.
[0137] Total cellular DNA from PS17 was digested with EcoRI and
separated by electrophoresis on a 0.8% (w/v) Agarose-TAE (50 mM
Tris-HCl, 20 mM NaOAc, 2.5 mM EDTA, pH=8.0) buffered gel. A
Southern blot of the gel was hybridized with a
[.sup.32P]-radiolabeled oligonucleotide probe derived from the
N-terminal amino acid sequence of purified 130 kDa protein from
PS17. The sequence of the oligonucleotide synthesized is
(GCAATTTTAAATGAATTATATCC) (SEQ ID NO. 24). Results showed that the
hybridizing EcoRI fragments of PS17 are 5.0 kb, 4.5 kb, 2.7 kb and
1.8 kb in size, presumptively identifying at least four new
nematode-active toxin genes, PS17d, PS17b, PS17a and PS17e,
respectively.
[0138] A library was constructed from PS17 total cellular DNA
partially digested with Sau3A and size fractionated by
electrophoresis. The 9 to 23 kb region of the gel was excised and
the DNA was electroeluted and then concentrated using an Elutip.TM.
ion exchange column (Schleicher and Schuel, Keene N.H.). The
isolated Sau3A fragments were ligated into LambdaGEM-11.TM.
(PROMEGA). The packaged phage were plated on KW251 E. coli cells
(PROMEGA) at a high titer and screened using the above radiolabeled
synthetic oligonucleotide as a nucleic acid hybridization probe.
Hybridizing plaques were purified and rescreened at a lower plaque
density. Single isolated purified plaques that hybridized with the
probe were used to infect KW251 E. coli cells in liquid culture for
preparation of phage for DNA isolation. DNA was isolated by
standard procedures.
[0139] Recovered recombinant phage DNA was digested with EcoRI and
separated by electrophoresis on a 0.8% agarose-TAE gel. The gel was
Southern blotted and hybridized with the oligonucleotide probe to
characterize the toxin genes isolated from the lambda library. Two
patterns were present, clones containing the 4.5 kb (PS17b) or the
2.7 kb (PS17a) EcoRI fragments. Preparative amounts of phage DNA
were digested with SalI (to release the inserted DNA from lambda
arms) and separated by electrophoresis on a 0.6% agarose-TAE gel.
The large fragments, electroeluted and concentrated as described
above, were ligated to SalI-digested and dephosphorylated pBClac,
an E. coli/B.t. shuttle vector comprised of replication origins
from pBC16 and pUC19. The ligation mix was introduced by
transformation into NM522 competent E. coli cells and plated on LB
agar containing ampicillin, isopropyl-(Beta)-D-thiogalactosi- de
(IPTG) and 5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL).
White colonies, with putative insertions in the
(Beta)-galactosidase gene of pBClac, were subjected to standard
rapid plasmid purification procedures to isolate the desired
plasmids. The selected plasmid containing the 2.7 kb EcoRI fragment
was named pMYC1627 and the plasmid containing the 4.5 kb EcoRI
fragment was called pMYC1628.
[0140] The toxin genes were sequenced by the standard Sanger
dideoxy chain termination method using the synthetic
oligonucleotide probe, disclosed above, and by "walking" with
primers made to the sequence of the new toxin genes.
[0141] The PS17 toxin genes were subcloned into the shuttle vector
pHT3101 (Lereclus, D. et al. [1989] FEMS Microbiol. Lett.
60:211-218) using standard methods for expression in B.t. Briefly,
SalI fragments containing the 17a and 17b toxin genes were isolated
from pMYC1629 and pMYC1627, respectively, by preparative agarose
gel electrophoresis, electroelution, and concentrated, as described
above. These concentrated fragments were ligated into SalI-cleaved
and dephosphorylated pHT3101. The ligation mixtures were used
separately to transform frozen, competent E. coli NM522. Plasmids
from each respective recombinant E. coli strain were prepared by
alkaline lysis and analyzed by agarose gel electrophoresis. The
resulting subclones, pMYC2311 and pMYC2309, harbored the 17a and
17b toxin genes, respectively. These plasmids were transformed into
the acrystalliferous B.t. strain, HD-1 cryB (Aronson, A., Purdue
University, West Lafayette, Ind.), by standard electroporation
techniques (Instruction Manual, Biorad, Richmond, Calif.).
[0142] Recombinant B.t. strains HD-1 cryB [pMYC2311] and [pMYC2309]
were grown to sporulation and the proteins purified by NaBr
gradient centrifugation as described above for the wild-type B.t.
proteins.
EXAMPLE 4
[0143] Activity of the B.t. Toxin Protein and Gene Product against
Caenorhabditis elegans
[0144] Caenorhabditis elegans (CE) was cultured as described by
Simpkin and Coles (J. Chem. Tech. Biotechnol. 31:66-69, 1981) in
coming (Corning Glass Works, Corning, N.Y.) 24-well tissue culture
plates containing 1 ml S-basal media, 0.5 mg ampicillin and 0.01 mg
cholesterol. Each well also contained ca. 10.sup.8 cells of
Escherichia coli strain OP-50, a uracil auxotroph. The wells were
seeded with ca. 100-200 CE per well and incubated at 20.degree. C.
Samples of protein (obtained from the wild type B.t. or the
recombinant B.t.) were added to the wells by serial dilution. Water
served as the control as well as the vehicle to introduce the
proteins to the wells.
[0145] Each of the wells were examined daily and representative
results are shown in Table 5 as follow:
11TABLE 5 .mu.g % Kill with protein from indicated isolate Toxin
HD-1 cryB [pMYC 2309] HD-1 cryB [pMYC 2311] PS17 100 25 50 75 32 25
50 75 10 50 25 50 1 0 0 0
EXAMPLE 5
[0146] Molecular Cloning of Gene Encoding a Novel Toxin from
Bacillus thuringiensis Strain PS52A1.
[0147] Total cellular DNA was prepared from Bacillus thuringiensis
PS52A1 (B.t. PS52A1) as disclosed in Example 3.
[0148] RFLP analyses were performed by standard hybridization of
Southern blots of PS52A1 DNA with a .sup.32P-labeled
oligonucleotide probe designed from the N-terminal amino acid
sequence disclosed in Example 2. The sequence of this probe is:
12 5' ATG ATT ATT GAT TCT AAA ACA ACA TTA CCA AGA CAT TCA/T TTA
ATA/T AAT (SEQ ID NO.25) ACA/T ATA/T AA 3'
[0149] This probe was designated 52A1-C. Hybridizing bands included
an approximately 3.6 kbp HindIII fragment and an approximately 8.6
kbp EcoRV fragment. A gene library was constructed from PS52A1 DNA
partially digested with Sau3A. Partial restriction digests were
fractionated by agarose gel electophoresis. DNA fragments 6.6 to 23
kbp in size were excised from the gel, electroeluted from the gel
slice, and recovered by ethanol precipitation after purification on
an Elutip-D ion exchange column. The Sau3A inserts were ligated
into BamHI-digested LambdaGem-11 (Promega). Recombinant by phage
were packaged and plated on E. coli KW251 cells (Promega). Plaques
were screened by hybridization with the radiolabeled 52A1-C
oligonucleotide probe disclosed above. Hybridizing phage were
plaque-purified and used to infect liquid cultures ofE. coli KW251
cells for isolation of phage DNA by standard procedures (Maniatis
et al.). For subeloning, preparative amounts of DNA were digested
with EcoRI and SalI, and electrophoresed on an agarose gel. The
approximately 3.1 kbp band containing the toxin gene was excised
from the gel, electroeluted from the gel slice, and purified by ion
exchange chromatography as above. The purified DNA insert was
ligated into EcoRI+SalI-digested pHTBlueII (an E. coli/B.
thuringiensis shuttle vector comprised of pBluescript S/K
[Stratagene] and the replication origin from a resident B.t.
plasmid [D. Lereclus et al. 1989. FEMS Microbiology Letters
60:211-218]). The ligation mix was used to transform frozen,
competent E. coli NM522 cells (ATCC 47000). Transformants were
plated on LB agar containing ampicillin,
isopropyl-(Beta)-D-thiogalactoside (IPTG), and
5-Bromo4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL). Plasmids
were purified from putative recombinants by alkaline lysis
(Maniatis et al.) and analyzed by electrophoresis of EcoRI and SalI
digests on agarose gels. The desired plasmid construct, pMYC2321
contains a toxin gene that is novel compared to the maps of other
toxin genes encoding nematicidal proteins.
[0150] Plasmid pMYC2321 was introduced into an acrystalliferous
(Cry.sup.-) B.t. host by electroporation. Expression of an
approximately 55-60 kDa crystal protein was verified by SDS-PAGE
analysis. NaBr-purified crystals were prepared as described in
Example 3 for determination of toxicity of the cloned gene product
to Pratylenchus spp.
EXAMPLE 6
[0151] Activity of the B.t. PS52A1 Toxin Protein and Gene Product
against the Root Lesion Nematode, Pratylenchus scribneri
[0152] Pratylenchus scribneri was reared aseptically on excised
corn roots in Gamborg's B5 medium (GIBCO Laboratories, Grand
Island, N.Y.). Bioassays were done in 24 well assay plates (Corning
#25820) using L 3-4 larvae as described by Tsai and Van Gundy (J.
Nematol. 22(3):327-332). Approximately 20 nematodes were placed in
each well. A total of 80-160 nematodes were used in each treatment.
Samples of protein were suspended in aqueous solution using a
hand-held homogenizer.
[0153] Mortality was assessed by prodding with a dull probe 7 days
after treatment. Larvae that did not respond to prodding were
considered moribund. Representative results are shown below.
13 Rate (ppm) Percent Moribund 200 75 Control 5
EXAMPLE 7
[0154] Molecular Cloning of Gene Encoding a Novel Toxin from
Bacillus Thuringiensis Strain PS69D1
[0155] Total cellular DNA was prepared from PS69D1 (B.t. PS69D1) as
disclosed in Example 3. RFLP analyses were performed by standard
hybridization of Southern blots of PS69D1 DNA with a 32P-labeled
oligonucleotide probe designated as 69D1-D. The sequence of the
69D1-D probe was:
[0156] 5'AAA CAT ATT AGA TTA GCA CAT ATT TTT GCA ACA CAA AA 3'(SEQ
ID NO. 26)
[0157] Hybridizing bands included an approximately 2.0 kbp HindIII
fragment.
[0158] A gene library was constructed from PS69D1 DNA partially
digested with Sau3A. Partial restriction digests were fractionated
by agarose gel electrophoresis. DNA fragments 6.6 to 23 kbp in size
were excised from the gel, electroeluted from the gel slice, and
recovered by ethanol precipitation after purification on an
Elutip-D ion exchange column. The Sau3A inserts were ligated into
BamHI-digested LambdaGem-11 (Promega, Madison, Wis.). Recombinant
phage were packaged and plated on E. coli KW251 cells (Promega,
Madison, Wis.). Plaques were screened by hybridization with the
radiolabeled 69D1-D oligonucleotide probe. Hybridizing phage were
plaque-purified and used to infect liquid cultures of E. coli KW251
cells for isolation of phage DNA by standard procedures (Maniatis
et al. [1982] Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor, N.Y.). For subcloning, preparative amounts of DNA were
digested with HindIII and electrophoresed on an agarose gel. The
approximately, 2.0 kbp band containing the toxin gene was excised
from the gel, electroeluted from the gel slice, and purified by ion
exchange chromatography as above. The purified DNA insert was
ligated into HindIII-digested pHTBlueII (and E. coli/B.t. shuttle
vector comprised of pBluescript S/K (Stratagene, San Diego, Calif.)
and the replication origin from a resident B.t. plasmid (D.
Lereclus et al [1989] FEMS Microbiol. Lett. 60:211-218). The
ligation mix was used to transform frozen, competent E. coli NM522
cells (ATCC 47000). Transformants were plated on LB agar containing
5-bromo-4-chloro-3-indolyl-(Beta)-D-galactos- ide (XGAL). Plasmids
were purified from putative recombinants by alkaline lysis
(Maniatis et al., supra) and analyzed by electrophoresis of HindIII
digests on agarose gels. The desired plasmid construct, pMYC2317,
contains a toxin gene that is novel compared to the maps ofother
toxin genes encoding insecticidal proteins.
EXAMPLE 8
[0159] Molecular Cloning of a Gene Encoding a Novel Toxin from
Bacillus thuringiensis Strain PS63B
[0160] Example 2 shows the aminoterminal and internal polypeptide
sequences of the PS63B toxin protein as determined by standard
Edman protein sequencing. From these sequences, two oligonucleotide
primers were designed using a codon frequency table assembled from
B.t. genes encoding .delta.-endotoxins. The sequence of the forward
primer (63B-A) was complementary to the predicted DNA sequence at
the 5' end of the gene:
[0161] 63B-A-5'CAA T/CTA CAA GCA/T CAA CC 3'(SEQ ID NO. 27)
[0162] The sequence of the reverse primer (63B-INT) was
complementary to the inverse of the internal predicted DNA
sequence:
[0163] 63B-INT-5'TTC ATC TAA AAT TCT TTG A/TAC 3'(SEQ ID NO.
28)
[0164] These primers were used in standard polymerase chain
reactions (Cetus Corporation) to amplify an approximately 460 bp
fragment of the 63B toxin gene for use as a DNA cloning probe.
Standard Southern blots oftotal cellular DNA from PS63B were
hybridized with the radiolabeled PCR probe. Hybridizing bands
included an approximately 4.4 kbp XbaI fragment, an approximately
2.0 kbp HindIII fragment, and an approximately 6.4 kbp SpeI
fragment.
[0165] Total cellular DNA was prepared from Bacillus thuringiensis
(B.t.) cells grown to an optical density of 1.0 at 600 nm. The
cells were recovered by centrifugation and protoplasts were
prepared in lysis mix (300 mM sucrose, 25 mM Tris-HCl, 25 mM EDTA,
pH=8.0) and lysozyme at a concentration of 20 mg/ml. The
protoplasts were ruptured by addition of ten volumes of 0.1 M NaCl,
0.1 M Tris-HCl pH 8.0, and 0.1% SDS. The cellular material was
quickly frozen at -70.degree. C. and thawed to 37.degree. C. twice.
The supernatant was extracted twice with phenol/chloroform (1:1).
The nucleic acids were precipitated with ethanol. To remove as much
RNA as possible from the DNA preparation, RNase at final
concentration of 200,.mu.g/ml was added. After incubation at
37.degree. C. for 1 hour, the solution was extracted once with
phenol/chloroform and precipitated with ethanol.
[0166] A gene library was constructed from PS63B total cellular DNA
partially digested with NdeII and size fractioned by gel
electrophoresis. The 9-23 kb region of the gel was excised and the
DNA was electroeluted and then concentrated using an Elutip-d ion
exchange column (Schleicher and Schuel, Keene, N.H.). The isolated
NdeII fragments were ligated into BamHI-digested LambdaGEM-11
(PROMEGA). The packaged phage were plated on E. coli KW251 cells
(PROMEGA) at a high titer and screened using the radiolabeled
approximately 430 bp fragment probe amplified with the 63B-A and
63B internal primers (SEQ ID NOS. 27 and 28, respectively) by
polymerase chain reaction. Hybridizing plaques were purified and
rescreened at a lower plaque density. Single isolated, purified
plaques that hybridized with the probe were used to infect KW251
cells in liquid culture for preparation of phage for DNA isolation.
DNA was isolated by standard procedures (Maniatis et al., supra).
Preparative amounts of DNA were digested with SalI (to release the
inserted DNA from lambda sequences) and separated by
electrophoresis on a 0.6% agarose-TAE gel. The large fragments were
purified by ion exchange chromatography as above and ligated to
SalI-digested, dephosphorylated pHTBlueII (an E. coli/B.t. shuttle
vector comprised of pBlueScript S/K [Stratagene, San Diego, Calif.]
and the replication origin from a resident B.t. plasmid [Lereclus,
D. et al. (1989) FEMS Microbiol. Lett. 60:211-218]). The ligation
mix was introduced by transformation into competent E. coli NM522
cells (ATCC 47000) and plated on LB agar containing ampicillin (100
.mu.g/ml), IPTG (2%), and XGAL (2%). White colonies, with putative
restriction fragment insertions in the (Beta)-galactosidase gene of
pHTBlueII, were subjected to standard rapid plasmid purification
procedures (Maniatis et al., supra). Plasmids ere analyzed by SalI
digestion and agarose gel electrophoresis. The desired plasmid
construct, pMYC1641, contains an approximately 14 kb SalI
insert.
[0167] For subdloning, preparative amounts of DNA were digested
with XbaI and electrophoresed on an agarose gel. The approximately
4.4 kbp band containing the toxin gene was excised from the gel,
electroeluted from the gel slice, and purified by ion exchange
chromatography as above. This fragment was ligated into XbaI cut
pHTBlueII and the resultant plasmid was designated pMYC1642.
EXAMPLE 9
[0168] Cloning of a Novel Toxin Gene from B.t. PS33F2 and
Transformation into Escherichia coli
[0169] Total cellular DNA was prepared from B.t. PS33F2 cells grown
to an optical density, at 600 nm, of 1.0. Cells were pelleted by
centrifugation and resuspended in protoplast buffer (20 mg/ml
lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA).
After incubation at 37.degree. C. for 1 hour, protoplasts were
lysed by the addition of nine volumes of a solution of 0.1 M NaCl,
0.1% SDS, 0.1 M Tris-Cl followed by two cycles of freezing and
thawing. The cleared lysate was extracted twice with
phenol:chloroform (1:1). Nucleic acids were precipitated with two
volumes of ethanol and pelleted by centrifugation. The pellet was
resuspended in 10 mM Tris-Cl, 1 mM EDTA (TE) and RNase was added to
a final concentration of 50 .mu.g/ml. After incubation at
37.degree. C. for 1 hour, the solution was extracted once each with
phenol:chloroforrn (1:1) and TE-saturated chloroform. DNA was
precipitated from the aqueous phase by the addition of one-tenth
volume of 3 M NaOAc and two volumes of ethanol. DNA was pelleted by
centrifugation, washed with 70% ethanol, dried, and resuspended in
TE.
[0170] Plasmid DNA was extracted from protoplasts prepared as
described above. Protoplasts were lysed by the addition of nine
volumes of a solution of 10 mM Tris-Cl, 1 mM EDTA, 0.085 N NaOH,
0.1% SDS, pH=8.0. SDS was added to 1% final concentration to
complete lysis. One-half volume of 3 M KOAc was then added and the
cellular material was precipitated overnight at 4.degree. C. After
centrifugation, the DNA was precipitated with ethanol and plasmids
were purified by isopycnic centrifugation on cesium
chloride-ethidium bromide gradients.
[0171] Restriction Fragment Length Polymorphism (RFLP) analyses
were performed by standard hybridization of Southern blots of
PS33F2 plasmid and total cellular DNA with the following two
.sup.32P-labelled oligonucleotide probes (designed to the
N-terminal amino acid sequence disclosed in Example 2):
14 Probe 33F2A: 5' GCA/T ACA/T TTA AAT GAA GTA/T TAT 3' (SEQ ID
NO.33) Probe 33F2B: 5' AAT GAA GTA/T TAT CCA/T GTA/T AAT 3' (SEQ ID
NO.34)
[0172] Hybridizing bands included an approximately 5.85 kbp EcoRI
fragment. Probe 33F2A and a reverse PCR primer were used to amplify
a DNA fragment of approximately 1.8 kbp for use as a hybridization
probe for cloning the PS33F2 toxin gene. The sequence of the
reverse primer was:
[0173] 5'GCAAGCGGCCGCTTATGGAATAAATTCAATT C/T T/G A/G TC T/A A
3'(SEQ ID NO. 35).
[0174] A gene library was constructed from PS33F2 plasmid DNA
digested withEcoRI. Restriction digests were fractionated by
agarose gel electrophoresis. DNA fragments 4.3-6.6 kbp were excised
from the gel, electroeluted from the gel slice, and recovered by
ethanol precipitation after purification on an Elutip-D ion
exchange column (Schleicher and Schuel, Keene N.H.). The EcoRI
inserts were ligated into EcoRI-digested pHTBlueII (an E. coli/B.
thuringiensis shuttle vector comprised of pBluescript S/K
[Stratagene] and the replication origin from a resident B.t.
plasmid [D. Lereclus et al. 1989. FEMS Microbial. Lett.
60:211-218]). The ligation mixture was transformed into frozen,
competent NM522 cells (ATCC 47000). Transformants were plated on LB
agar containing ampicillin, isopropyl-(Beta)-D-thiogalactoside
(IPTG), and 5-bromo-4-chloro-3-indolyl-(Beta)-D-galactoside (XGAL).
Colonies were screened by hybridization with the radiolabeled PCR
amplified probe described above. Plasmids were purified from
putative toxin gene clones by alkaline lysis and analyzed by
agarose gel electrophoresis of restriction digests. The desired
plasmid construct, pMYC2316, contains an approximately 5.85 kbp
Eco4RI insert; the toxin gene residing on this DNA fragment (33F2a)
is novel compared to the DNA sequences of other toxin genes
encoding nematicidal proteins.
[0175] Plasmid pMYC2316 was introduced into the acrystalliferous
(Cry-) B.t. host, HD-1 CryB (A. Aronson, Purdue University, West
Lafayette, Ind.) by electroporation. Expression of an approximately
120-140 kDa crystal protein was verified by SDS-PAGE analysis.
Crystals were purified on NaBr gradients (M. A. Pfannenstiel et al.
1984. FEMS Microbiol. Lett. 21:39) for determination of toxicity of
the cloned gene product to Pratylenchus spp.
EXAMPLE 10
[0176] Activity of the B.t. Gene Product 33F2 against the Plant
Nematode Pratylenchus spp.
[0177] Pratylenchus spp. was reared aseptically on excised corn
roots in Gamborg's B5 medium (GIBCO.RTM. Laboratories, Grand
Island, N.Y.) Bioassays were done in 24 well assay plates (Corning
#25820) using L 3-4 larvae as described by Tsai and van Gundy (J.
Nematol. 22(3):327-332). Approximately 20 nematodes were placed in
each well. A total of 80-160 nematodes were used in each treatment.
Samples of protein were suspended in an aqueous solution using a
hand-held Dounce homogenizer.
[0178] Mortality was assessed visually 3 days after treatment.
Larvae that were nearly straight and not moving were considered
moribund. Representative results are as follows:
15 33F2a (ppm) % Moribund 0 12 75 78
[0179] Species of Pratylenchus, for example P. scribneri, are known
pathogens of many economically important crops including corn,
peanuts, soybean, alfalfa, beans, tomato, and citrus. These "root
lesion" nematodes are the second most economically damaging genus
of plant parasitic nematodes (after Meloidogyne--the "root knot"
nematode), and typify the migratory endoparasites.
EXAMPLE 11
[0180] Cloning of Novel Nematode-Active Genes Using Generic
Oligonucleotide Primers
[0181] The nematicidal gene of a new nematicidal B.t. can be
obtained from DNA of the strain by performing the standard
polymerase chain reaction procedure as in Example 8 using the
oligonucleotides of SEQ ID NO. 32 or SEQ ID NO. 30 as reverse
primers and SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 24, Probe B of
SEQ ID NO. 5 (AAT GAA GTA/T TAT CCA/T GTA/T AAT), or SEQ ID NO. 27
as forward primers. The expected PCR fragments would be
approximately 330 to 600 bp (with either reverse primer and SEQ ID
NO. 14), 1000 to 1400 bp (with either reverse primer and SEQ ID NO.
16), and 1800 to 2100 bp (with either reverse primer and any of the
three N-terminal primers, SEQ ID NO. 5 (Probe B), SEQ ID NO. 24,
and SEQ ID NO. 27). Alternatively, a complement from the primer
family described by SEQ ID NO. 14 can be used as reverse primer
with SEQ ID NO. 16, SEQ ID NO. 24, SEQ ID NO. 5 (Probe B), or SEQ
ID NO. 27 as forward primers. The expected PCR fragments would be
approximately 650 to 1000 bp with SEQ ID NO. 16, and 1400 to 1800
bp (for the three N-terminal primers, SEQ ID NO. 5 (Probe B), SEQ
ID NO. 24, and SEQ ID NO. 27). Amplified DNA fragments of the
indicated sizes can be radiolabeled and used as probes to clone the
entire gene as in Example 8.
EXAMPLE 12
[0182] Further Cloning of Novel Nematode-Active Genes Using Generic
Oligonucleotide Primers
[0183] A gene coding for a nematicidal toxin a new nematicidal B.t.
isolate can also be obtained from DNA of the strain by performing
the standard polymerase chain reaction procedure as in Example 8
using oligonucleotides derived from the PS52A1 and PS69D1 gene
sequences as follows:
[0184] 1. Forward primer "TGATTTT(T or A)(C or A)TCAATTATAT(A or
G)A(G or T)GTTTAT" (SEQ ID NO. 36) can be used with primers
complementary to probe "AAGAGTTA(C or T)TA(A or G)A(G or A)AAAGTA"
(SEQ ID NO. 37), probe "TTAGGACCATT(A or G)(C or T)T(T or
A)GGATTTGTTGT(A or T)TATGAAAT" (SEQ ID NO. 38), and probe "GA(C or
T)AGAGATGT(A or T)AAAAT(C or T)(T or A)TAGGAATG" (SEQ ID NO. 39) to
produce amplified fragments of approximately 440, 540, and 650 bp,
respectively.
[0185] 2. Forward primer "TT(A or C)TTAAA(A or T)C(A or
T)GCTAATGATATT" (SEQ ID NO. 40) can be used with primers
complementary to SEQ ID NO. 37, SEQ ID NO. 38, and SEQ ID NO. 39 to
produce amplified fragments of approximately 360, 460, and 570 bp,
respectively.
[0186] 3. Forward primer SEQ ID NO. 37 can be used with primers
complementary to SEQ ID NO. 38 and SEQ ID NO. 39 to produce
amplified fragments of approximately 100 and 215 bp,
respectively.
[0187] Amplified DNA fragments of the indicated sizes can be
radiolabeled and used as probes to clone the entire gene as in
Example 8.
EXAMPLE 13
[0188] Insertion of Toxin Gene into Plants
[0189] One aspect of the subject invention is the transformation of
plants with genes coding for a nematicidal toxin. The transformed
plants are resistant to attack by nematodes.
[0190] Genes coding for nematicidal toxins, as disclosed herein,
can be inserted into plant cells using a variety of techniques
which are well known in the art. For example, a large number of
cloning vectors comprising a replication system in E. coli and a
marker that permits selection of the transformed cells are
available for preparation for the insertion of foreign genes into
higher plants. The vectors comprise, for example, pBR322, pUC
series, M13mp series, pACYC184, etc. Accordingly, the sequence
coding for the B.t. toxin can be inserted into the vector at a
suitable restriction site. The resulting plasmid is used for
transformation into E. coli. The E. coli cells are cultivated in a
suitable nutrient medium, then harvested and lysed. The plasmid is
recovered. Sequence analysis, restriction analysis,
electrophoresis, and other biochemical-molecular biological methods
are generally carried out as methods of analysis. After each
manipulation, the DNA sequence used can be cleaved and joined to
the next DNA sequence. Each plasmid sequence can be cloned in the
same or other plasmids. Depending on the method of inserting
desired genes into the plant, other DNA sequences may be necessary.
If, for example, the Ti or Ri plasmid is used for the
transformation of the plant cell, then at least the right border,
but often the right and the left border of the Ti or Ri plasmid
T-DNA, has to be joined as the flanking region of the genes to be
inserted.
[0191] The use of T-DNA for the transformation of plant cells has
been intensively researched and sufficiently described in EP 120
516; Hoekema (1985) In: The Binary Plant Vector System,
Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et
al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J.
4:277-287.
[0192] Once the inserted DNA has been integrated in the genome, it
is relatively stable there and, as a rule, does not come out again.
It normally contains a selection marker that confers on the
transformed plant cells resistance to a biocide or an antibiotic,
such as kanamycin, G 418, bleomycin, hygromycin, or
chloramphenicol, inter alia. The individually employed marker
should accordingly permit the selection of transformed cells rather
than cells that do not contain the inserted DNA.
[0193] A large number of techniques are available for inserting DNA
into a plant host cell. Those techniques include transformation
with T-DNA using Agrobacterium tumefaciens or Agrobacterium
rhizogenes as transformation agent, fusion, injection, or
electroporation as well as other possible methods. If agrobacteria
are used for the transformation, the DNA to be inserted has to be
cloned into special plasmids, namely either into an intermediate
vector or into a binary vector. The intermediate vectors can be
integrated into the Ti or Ri plasmid by homologous recombination
owing to sequences that are homologous to sequences in the T-DNA.
The Ti or Ri plasmid also comprises the vir region necessary for
the transfer of the T-DNA. Intermediate vectors cannot replicate
themselves in agrobacteria. The intermediate vector can be
transferred into Agrobacterium tumefaciens by means of a helper
plasmid (conjugation). Binary vectors can replicate themselves both
in E. coli and in agrobacteria. They comprise a selection marker
gene and a linker or polylinker which are framed by the right and
left T-DNA border regions. They can be transformed directly into
agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181-187).
The agrobacterium used as host cell is to comprise a plasmid
carrying a vir region. The vir region is necessary for the transfer
of the T-DNA into the plant cell. Additional T-DNA may be
contained. The bacterium so transformed is used for the
transformation of plant cells. Plant explants can advantageously be
cultivated with Agrobacterium tumefaciens or Agrobacterium
rhizogenes for the transfer of the DNA into the plant cell. Whole
plants can then be regenerated from the infected plant material
(for example, pieces of leaf, segments of stalk, roots, but also
protoplasts or suspension-cultivated cells) in a suitable medium,
which may contain antibiotics or biocides for selection. The plants
so obtained can then be tested for the presence of the inserted
DNA. No special demands are made of the plasmids in the case of
injection and electroporarion. It is possible to use ordinary
plasmids, such as, for example, pUC derivatives.
[0194] The transformed cells grow inside the plants in the usual
manner. They can form germ cells and transmit the transformed
trait(s) to progeny plants. Such plants can be grown in the normal
manner and crossed with plants that have the same transformed
hereditary factors or other hereditary factors. The resulting
hybrid indivuals have the corresponding phenotypic properties.
[0195] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims.
Sequence CWU 1
1
40 1 4155 DNA Bacillus thuringiensis 1 atggcaattt taaatgaatt
atatccatct gtaccttata atgtattggc gtatacgcca 60 ccctcttttt
tacctgatgc gggtacacaa gctacacctg ctgacttaac agcttatgaa 120
caattgttga aaaatttaga aaaagggata aatgctggaa cttattcgaa agcaatagct
180 gatgtactta aaggtatttt tatagatgat acaataaatt atcaaacata
tgtaaatatt 240 ggtttaagtt taattacatt agctgtaccg gaaattggta
tttttacacc tttcatcggt 300 ttgttttttg ctgcattgaa taaacatgat
gctccacctc ctcctaatgc aaaagatata 360 tttgaggcta tgaaaccagc
gattcaagag atgattgata gaactttaac tgcggatgag 420 caaacatttt
taaatgggga aataagtggt ttacaaaatt tagcagcaag ataccagtct 480
acaatggatg atattcaaag ccatggagga tttaataagg tagattctgg attaattaaa
540 aagtttacag atgaggtact atctttaaat agtttttata cagatcgttt
acctgtattt 600 attacagata atacagcgga tcgaactttg ttaggtcttc
cttattatgc tatacttgcg 660 agcatgcatc ttatgttatt aagagatatc
attactaagg gtccgacatg ggattctaaa 720 attaatttca caccagatgc
aattgattcc tttaaaaccg atattaaaaa taatataaag 780 ctttactcta
aaactattta tgacgtattt cagaagggac ttgcttcata cggaacgcct 840
tctgatttag agtcctttgc aaaaaaacaa aaatatattg aaattatgac aacacattgt
900 ttagattttg caagattgtt tcctactttt gatccagatc tttatccaac
aggatcaggt 960 gatataagtt tacaaaaaac acgtagaatt ctttctcctt
ttatccctat acgtactgca 1020 gatgggttaa cattaaataa tacttcaatt
gatacttcaa attggcctaa ttatgaaaat 1080 gggaatggcg cgtttccaaa
cccaaaagaa agaatattaa aacaattcaa actgtatcct 1140 agttggagag
cgggacagta cggtgggctt ttacaacctt atttatgggc aatagaagtc 1200
caagattctg tagagactcg tttgtatggg cagcttccag ctgtagatcc acaggcaggg
1260 cctaattatg tttccataga ttcttctaat ccaatcatac aaataaatat
ggatacttgg 1320 aaaacaccac cacaaggtgc gagtgggtgg aatacaaatt
taatgagagg aagtgtaagc 1380 gggttaagtt ttttacaacg agatggtacg
agacttagtg ctggtatggg tggtggtttt 1440 gctgatacaa tatatagtct
ccctgcaact cattatcttt cttatctcta tggaactcct 1500 tatcaaactt
ctgataacta ttctggtcac gttggtgcat tggtaggtgt gagtacgcct 1560
caagaggcta ctcttcctaa tattataggt caaccagatg aacagggaaa tgtatctaca
1620 atgggatttc cgtttgaaaa agcttcttat ggaggtacag ttgttaaaga
atggttaaat 1680 ggtgcgaatg cgatgaagct ttctcctggg caatctatag
gtattcctat tacaaatgta 1740 acaagtggag aatatcaaat tcgttgtcgt
tatgcaagta atgataatac taacgttttc 1800 tttaatgtag atactggtgg
agcaaatcca attttccaac agataaactt tgcatctact 1860 gtagataata
atacgggagt acaaggagca aatggtgtct atgtagtcaa atctattgct 1920
acaactgata attcttttac agaaattcct gcgaagacga ttaatgttca tttaaccaac
1980 caaggttctt ctgatgtctt tttagaccgt attgaattta tacctttttc
tctacctctt 2040 atatatcatg gaagttataa tacttcatca ggtgcagatg
atgttttatg gtcttcttca 2100 aatatgaatt actacgatat aatagtaaat
ggtcaggcca atagtagtag tatcgctagt 2160 tctatgcatt tgcttaataa
aggaaaagtg ataaaaacaa ttgatattcc agggcattcg 2220 gaaaccttct
ttgctacgtt cccagttcca gaaggattta atgaagttag aattcttgct 2280
ggccttccag aagttagtgg aaatattacc gtacaatcta ataatccgcc tcaacctagt
2340 aataatggtg gtggtgatgg tggtggtaat ggtggtggtg atggtggtca
atacaatttt 2400 tctttaagcg gatctgatca tacgactatt tatcatggaa
aacttgaaac tgggattcat 2460 gtacaaggta attataccta tacaggtact
cccgtattaa tactgaatgc ttacagaaat 2520 aatactgtag tatcaagcat
tccagtatat tctccttttg atataactat acagacagaa 2580 gctgatagcc
ttgagcttga actacaacct agatatggtt ttgccacagt gaatggtact 2640
gcaacagtaa aaagtcctaa tgtaaattac gatagatcat ttaaactccc aatagactta
2700 caaaatatca caacacaagt aaatgcatta ttcgcatctg gaacacaaaa
tatgcttgct 2760 cataatgtaa gtgatcatga tattgaagaa gttgtattaa
aagtggatgc cttatcagat 2820 gaagtatttg gagatgagaa gaaggcttta
cgtaaattgg tgaatcaagc aaaacgtttg 2880 agtagagcaa gaaatcttct
gataggtggg agttttgaaa attgggatgc atggtataaa 2940 ggaagaaatg
tagtaactgt atctgatcat gaactattta agagtgatca tgtattatta 3000
ccaccaccag gattgtctcc atcttatatt ttccaaaaag tggaggaatc taaattaaaa
3060 ccaaatacac gttatattgt ttctggattc atcgcacatg gaaaagacct
agaaattgtt 3120 gtttcacgtt atgggcaaga agtgcaaaag gtcgtgcaag
ttccttatgg agaagcattc 3180 ccgttaacat caaatggacc agtttgttgt
cccccacgtt ctacaagtaa tggaacctta 3240 ggagatccac atttctttag
ttacagtatc gatgtaggtg cactagattt acaagcaaac 3300 cctggtattg
aatttggtct tcgtattgta aatccaactg gaatggcacg cgtaagcaat 3360
ttggaaattc gtgaagatcg tccattagca gcaaatgaaa tacgacaagt acaacgtgtc
3420 gcaagaaatt ggagaaccga gtatgagaaa gaacgtgcgg aagtaacaag
tttaattcaa 3480 cctgttatca atcgaatcaa cggattgtat gaaaatggaa
attggaacgg ttctattcgt 3540 tcagatattt cgtatcagaa tatagacgcg
attgtattac caacgttacc aaagttacgc 3600 cattggttta tgtcagatag
attcagtgaa caaggagata taatggctaa attccaaggt 3660 gcattaaatc
gtgcgtatgc acaactggaa caaagtacgc ttctgcataa tggtcatttt 3720
acaaaagatg cagctaattg gacaatagaa ggcgatgcac atcagataac actagaagat
3780 ggtagacgtg tattgcgact tccagattgg tcttcgagtg tatctcaaat
gattgaaatc 3840 gagaatttta atccagataa agaatacaac ttagtattcc
atgggcaagg agaaggaacg 3900 gttacgttgg agcatggaga agaaacaaaa
tatatagaaa cgcatacaca tcattttgcg 3960 aattttacaa cttctcaacg
tcaaggactc acgtttgaat caaataaagt gacagtgacc 4020 atttcttcag
aagatggaga attcttagtg gataatattg cgcttgtgga agctcctctt 4080
cctacagatg accaaaattc tgagggaaat acggcttcca gtacgaatag cgatacaagt
4140 atgaacaaca atcaa 4155 2 1385 PRT Bacillus thuringiensis 2 Met
Ala Ile Leu Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val Leu 1 5 10
15 Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp Ala Gly Thr Gln Ala Thr
20 25 30 Pro Ala Asp Leu Thr Ala Tyr Glu Gln Leu Leu Lys Asn Leu
Glu Lys 35 40 45 Gly Ile Asn Ala Gly Thr Tyr Ser Lys Ala Ile Ala
Asp Val Leu Lys 50 55 60 Gly Ile Phe Ile Asp Asp Thr Ile Asn Tyr
Gln Thr Tyr Val Asn Ile 65 70 75 80 Gly Leu Ser Leu Ile Thr Leu Ala
Val Pro Glu Ile Gly Ile Phe Thr 85 90 95 Pro Phe Ile Gly Leu Phe
Phe Ala Ala Leu Asn Lys His Asp Ala Pro 100 105 110 Pro Pro Pro Asn
Ala Lys Asp Ile Phe Glu Ala Met Lys Pro Ala Ile 115 120 125 Gln Glu
Met Ile Asp Arg Thr Leu Thr Ala Asp Glu Gln Thr Phe Leu 130 135 140
Asn Gly Glu Ile Ser Gly Leu Gln Asn Leu Ala Ala Arg Tyr Gln Ser 145
150 155 160 Thr Met Asp Asp Ile Gln Ser His Gly Gly Phe Asn Lys Val
Asp Ser 165 170 175 Gly Leu Ile Lys Lys Phe Thr Asp Glu Val Leu Ser
Leu Asn Ser Phe 180 185 190 Tyr Thr Asp Arg Leu Pro Val Phe Ile Thr
Asp Asn Thr Ala Asp Arg 195 200 205 Thr Leu Leu Gly Leu Pro Tyr Tyr
Ala Ile Leu Ala Ser Met His Leu 210 215 220 Met Leu Leu Arg Asp Ile
Ile Thr Lys Gly Pro Thr Trp Asp Ser Lys 225 230 235 240 Ile Asn Phe
Thr Pro Asp Ala Ile Asp Ser Phe Lys Thr Asp Ile Lys 245 250 255 Asn
Asn Ile Lys Leu Tyr Ser Lys Thr Ile Tyr Asp Val Phe Gln Lys 260 265
270 Gly Leu Ala Ser Tyr Gly Thr Pro Ser Asp Leu Glu Ser Phe Ala Lys
275 280 285 Lys Gln Lys Tyr Ile Glu Ile Met Thr Thr His Cys Leu Asp
Phe Ala 290 295 300 Arg Leu Phe Pro Thr Phe Asp Pro Asp Leu Tyr Pro
Thr Gly Ser Gly 305 310 315 320 Asp Ile Ser Leu Gln Lys Thr Arg Arg
Ile Leu Ser Pro Phe Ile Pro 325 330 335 Ile Arg Thr Ala Asp Gly Leu
Thr Leu Asn Asn Thr Ser Ile Asp Thr 340 345 350 Ser Asn Trp Pro Asn
Tyr Glu Asn Gly Asn Gly Ala Phe Pro Asn Pro 355 360 365 Lys Glu Arg
Ile Leu Lys Gln Phe Lys Leu Tyr Pro Ser Trp Arg Ala 370 375 380 Gly
Gln Tyr Gly Gly Leu Leu Gln Pro Tyr Leu Trp Ala Ile Glu Val 385 390
395 400 Gln Asp Ser Val Glu Thr Arg Leu Tyr Gly Gln Leu Pro Ala Val
Asp 405 410 415 Pro Gln Ala Gly Pro Asn Tyr Val Ser Ile Asp Ser Ser
Asn Pro Ile 420 425 430 Ile Gln Ile Asn Met Asp Thr Trp Lys Thr Pro
Pro Gln Gly Ala Ser 435 440 445 Gly Trp Asn Thr Asn Leu Met Arg Gly
Ser Val Ser Gly Leu Ser Phe 450 455 460 Leu Gln Arg Asp Gly Thr Arg
Leu Ser Ala Gly Met Gly Gly Gly Phe 465 470 475 480 Ala Asp Thr Ile
Tyr Ser Leu Pro Ala Thr His Tyr Leu Ser Tyr Leu 485 490 495 Tyr Gly
Thr Pro Tyr Gln Thr Ser Asp Asn Tyr Ser Gly His Val Gly 500 505 510
Ala Leu Val Gly Val Ser Thr Pro Gln Glu Ala Thr Leu Pro Asn Ile 515
520 525 Ile Gly Gln Pro Asp Glu Gln Gly Asn Val Ser Thr Met Gly Phe
Pro 530 535 540 Phe Glu Lys Ala Ser Tyr Gly Gly Thr Val Val Lys Glu
Trp Leu Asn 545 550 555 560 Gly Ala Asn Ala Met Lys Leu Ser Pro Gly
Gln Ser Ile Gly Ile Pro 565 570 575 Ile Thr Asn Val Thr Ser Gly Glu
Tyr Gln Ile Arg Cys Arg Tyr Ala 580 585 590 Ser Asn Asp Asn Thr Asn
Val Phe Phe Asn Val Asp Thr Gly Gly Ala 595 600 605 Asn Pro Ile Phe
Gln Gln Ile Asn Phe Ala Ser Thr Val Asp Asn Asn 610 615 620 Thr Gly
Val Gln Gly Ala Asn Gly Val Tyr Val Val Lys Ser Ile Ala 625 630 635
640 Thr Thr Asp Asn Ser Phe Thr Glu Ile Pro Ala Lys Thr Ile Asn Val
645 650 655 His Leu Thr Asn Gln Gly Ser Ser Asp Val Phe Leu Asp Arg
Ile Glu 660 665 670 Phe Ile Pro Phe Ser Leu Pro Leu Ile Tyr His Gly
Ser Tyr Asn Thr 675 680 685 Ser Ser Gly Ala Asp Asp Val Leu Trp Ser
Ser Ser Asn Met Asn Tyr 690 695 700 Tyr Asp Ile Ile Val Asn Gly Gln
Ala Asn Ser Ser Ser Ile Ala Ser 705 710 715 720 Ser Met His Leu Leu
Asn Lys Gly Lys Val Ile Lys Thr Ile Asp Ile 725 730 735 Pro Gly His
Ser Glu Thr Phe Phe Ala Thr Phe Pro Val Pro Glu Gly 740 745 750 Phe
Asn Glu Val Arg Ile Leu Ala Gly Leu Pro Glu Val Ser Gly Asn 755 760
765 Ile Thr Val Gln Ser Asn Asn Pro Pro Gln Pro Ser Asn Asn Gly Gly
770 775 780 Gly Asp Gly Gly Gly Asn Gly Gly Gly Asp Gly Gly Gln Tyr
Asn Phe 785 790 795 800 Ser Leu Ser Gly Ser Asp His Thr Thr Ile Tyr
His Gly Lys Leu Glu 805 810 815 Thr Gly Ile His Val Gln Gly Asn Tyr
Thr Tyr Thr Gly Thr Pro Val 820 825 830 Leu Ile Leu Asn Ala Tyr Arg
Asn Asn Thr Val Val Ser Ser Ile Pro 835 840 845 Val Tyr Ser Pro Phe
Asp Ile Thr Ile Gln Thr Glu Ala Asp Ser Leu 850 855 860 Glu Leu Glu
Leu Gln Pro Arg Tyr Gly Phe Ala Thr Val Asn Gly Thr 865 870 875 880
Ala Thr Val Lys Ser Pro Asn Val Asn Tyr Asp Arg Ser Phe Lys Leu 885
890 895 Pro Ile Asp Leu Gln Asn Ile Thr Thr Gln Val Asn Ala Leu Phe
Ala 900 905 910 Ser Gly Thr Gln Asn Met Leu Ala His Asn Val Ser Asp
His Asp Ile 915 920 925 Glu Glu Val Val Leu Lys Val Asp Ala Leu Ser
Asp Glu Val Phe Gly 930 935 940 Asp Glu Lys Lys Ala Leu Arg Lys Leu
Val Asn Gln Ala Lys Arg Leu 945 950 955 960 Ser Arg Ala Arg Asn Leu
Leu Ile Gly Gly Ser Phe Glu Asn Trp Asp 965 970 975 Ala Trp Tyr Lys
Gly Arg Asn Val Val Thr Val Ser Asp His Glu Leu 980 985 990 Phe Lys
Ser Asp His Val Leu Leu Pro Pro Pro Gly Leu Ser Pro Ser 995 1000
1005 Tyr Ile Phe Gln Lys Val Glu Glu Ser Lys Leu Lys Pro Asn Thr
1010 1015 1020 Arg Tyr Ile Val Ser Gly Phe Ile Ala His Gly Lys Asp
Leu Glu 1025 1030 1035 Ile Val Val Ser Arg Tyr Gly Gln Glu Val Gln
Lys Val Val Gln 1040 1045 1050 Val Pro Tyr Gly Glu Ala Phe Pro Leu
Thr Ser Asn Gly Pro Val 1055 1060 1065 Cys Cys Pro Pro Arg Ser Thr
Ser Asn Gly Thr Leu Gly Asp Pro 1070 1075 1080 His Phe Phe Ser Tyr
Ser Ile Asp Val Gly Ala Leu Asp Leu Gln 1085 1090 1095 Ala Asn Pro
Gly Ile Glu Phe Gly Leu Arg Ile Val Asn Pro Thr 1100 1105 1110 Gly
Met Ala Arg Val Ser Asn Leu Glu Ile Arg Glu Asp Arg Pro 1115 1120
1125 Leu Ala Ala Asn Glu Ile Arg Gln Val Gln Arg Val Ala Arg Asn
1130 1135 1140 Trp Arg Thr Glu Tyr Glu Lys Glu Arg Ala Glu Val Thr
Ser Leu 1145 1150 1155 Ile Gln Pro Val Ile Asn Arg Ile Asn Gly Leu
Tyr Glu Asn Gly 1160 1165 1170 Asn Trp Asn Gly Ser Ile Arg Ser Asp
Ile Ser Tyr Gln Asn Ile 1175 1180 1185 Asp Ala Ile Val Leu Pro Thr
Leu Pro Lys Leu Arg His Trp Phe 1190 1195 1200 Met Ser Asp Arg Phe
Ser Glu Gln Gly Asp Ile Met Ala Lys Phe 1205 1210 1215 Gln Gly Ala
Leu Asn Arg Ala Tyr Ala Gln Leu Glu Gln Ser Thr 1220 1225 1230 Leu
Leu His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr 1235 1240
1245 Ile Glu Gly Asp Ala His Gln Ile Thr Leu Glu Asp Gly Arg Arg
1250 1255 1260 Val Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gln
Met Ile 1265 1270 1275 Glu Ile Glu Asn Phe Asn Pro Asp Lys Glu Tyr
Asn Leu Val Phe 1280 1285 1290 His Gly Gln Gly Glu Gly Thr Val Thr
Leu Glu His Gly Glu Glu 1295 1300 1305 Thr Lys Tyr Ile Glu Thr His
Thr His His Phe Ala Asn Phe Thr 1310 1315 1320 Thr Ser Gln Arg Gln
Gly Leu Thr Phe Glu Ser Asn Lys Val Thr 1325 1330 1335 Val Thr Ile
Ser Ser Glu Asp Gly Glu Phe Leu Val Asp Asn Ile 1340 1345 1350 Ala
Leu Val Glu Ala Pro Leu Pro Thr Asp Asp Gln Asn Ser Glu 1355 1360
1365 Gly Asn Thr Ala Ser Ser Thr Asn Ser Asp Thr Ser Met Asn Asn
1370 1375 1380 Asn Gln 1385 3 3867 DNA Bacillus thuringiensis 3
atggcaattt taaatgaatt atatccatct gtaccttata atgtattggc gtatacgcca
60 ccctcttttt tacctgatgc gggtacacaa gctacacctg ctgacttaac
agcttatgaa 120 caattgttga aaaatttaga aaaagggata aatgctggaa
cttattcgaa agcaatagct 180 gatgtactta aaggtatttt tatagatgat
acaataaatt atcaaacata tgtaaatatt 240 ggtttaagtt taattacatt
agctgtaccg gaaattggta tttttacacc tttcatcggt 300 ttgttttttg
ctgcattgaa taaacatgat gctccacctc ctcctaatgc aaaagatata 360
tttgaggcta tgaaaccagc gattcaagag atgattgata gaactttaac tgcggatgag
420 caaacatttt taaatgggga aataagtggt ttacaaaatt tagcagcaag
ataccagtct 480 acaatggatg atattcaaag ccatggagga tttaataagg
tagattctgg attaattaaa 540 aagtttacag atgaggtact atctttaaat
agtttttata cagatcgttt acctgtattt 600 attacagata atacagcgga
tcgaactttg ttaggtcttc cttattatgc tatacttgcg 660 agcatgcatc
ttatgttatt aagagatatc attactaagg gtccgacatg ggattctaaa 720
attaatttca caccagatgc aattgattcc tttaaaaccg atattaaaaa taatataaag
780 ctttactcta aaactattta tgacgtattt cagaagggac ttgcttcata
cggaacgcct 840 tctgatttag agtcctttgc aaaaaaacaa aaatatattg
aaattatgac aacacattgt 900 ttagattttg caagattgtt tcctactttt
gatccagatc tttatccaac aggatcaggt 960 gatataagtt tacaaaaaac
acgtagaatt ctttctcctt ttatccctat acgtactgca 1020 gatgggttaa
cattaaataa tacttcaatt gatacttcaa attggcctaa ttatgaaaat 1080
gggaatggcg cgtttccaaa cccaaaagaa agaatattaa aacaattcaa actgtatcct
1140 agttggagag cggcacagta cggtgggctt ttacaacctt atttatgggc
aatagaagtc 1200 caagattctg tagagactcg tttgtatggg cagcttccag
ctgtagatcc acaggcaggg 1260 cctaattatg tttccataga ttcttctaat
ccaatcatac aaataaatat ggatacttgg 1320 aaaacaccac cacaaggtgc
gagtgggtgg aatacaaatt taatgagagg aagtgtaagc 1380 gggttaagtt
ttttacaacg agatggtacg agacttagtg ctggtatggg tggtggtttt 1440
gctgatacaa tatatagtct ccctgcaact cattatcttt cttatctcta tggaactcct
1500 tatcaaactt ctgataacta ttctggtcac gttggtgcat tggtaggtgt
gagtacgcct 1560 caagaggcta ctcttcctaa tattataggt caaccagatg
aacagggaaa tgtatctaca 1620 atgggatttc cgtttgaaaa agcttcttat
ggaggtacag ttgttaaaga atggttaaat 1680 ggtgcgaatg cgatgaagct
ttctcctggg caatctatag gtattcctat tacaaatgta 1740 acaagtggag
aatatcaaat tcgttgtcgt tatgcaagta atgataatac taacgttttc 1800
tttaatgtag atactggtgg agcaaatcca attttccaac agataaactt tgcatctact
1860 gtagataata atacgggagt acaaggagca aatggtgtct atgtagtcaa
atctattgct 1920 acaactgata attcttttac agtaaaaatt cctgcgaaga
cgattaatgt tcatttaacc 1980 aaccaaggtt cttctgatgt ctttttagat
cgtattgagt ttgttccaat tctagaatca 2040 aatactgtaa ctatattcaa
caattcatat actacaggtt cagcaaatct tataccagca 2100 atagctcctc
tttggagtac tagttcagat aaagccctta caggttctat gtcaataaca 2160
ggtcgaacta cccctaacag tgatgatgct ttgcttcgat tttttaaaac
taattatgat 2220 acacaaacca ttcctattcc gggttccgga aaagatttta
caaatactct agaaatacaa 2280 gacatagttt ctattgatat ttttgtcgga
tctggtctac atggatccga tggatctata 2340 aaattagatt ttaccaataa
taatagtggt agtggtggct ctccaaagag tttcaccgag 2400 caaaatgatt
tagagaatat cacaacacaa gtgaatgctc tattcacatc taatacacaa 2460
gatgcacttg caacagatgt gagtgatcat gatattgaag aagtggttct aaaagtagat
2520 gcattatctg atgaagtgtt tggaaaagag aaaaaaacat tgcgtaaatt
tgtaaatcaa 2580 gcgaagcgct taagcaaggc gcgtaatctc ctggtaggag
gcaattttga taacttggat 2640 gcttggtata gaggaagaaa tgtagtaaac
gtatctaatc acgaactgtt gaagagtgat 2700 catgtattat taccaccacc
aggattgtct ccatcttata ttttccaaaa agtggaggaa 2760 tctaaattaa
aacgaaatac acgttatacg gtttctggat ttattgcgca tgcaacagat 2820
ttagaaattg tggtttctcg ttatgggcaa gaaataaaga aagtggtgca agttccttat
2880 ggagaagcat tcccattaac atcaagtgga ccagtttgtt gtatcccaca
ttctacaagt 2940 aatggaactt taggcaatcc acatttcttt agttacagta
ttgatgtagg tgcattagat 3000 gtagacacaa accctggtat tgaattcggt
cttcgtattg taaatccaac tggaatggca 3060 cgcgtaagca atttggaaat
tcgtgaagat cgtccattag cagcaaatga aatacgacaa 3120 gtacaacgtg
tcgcaagaaa ttggagaacc gagtatgaga aagaacgtgc ggaagtaaca 3180
agtttaattc aacctgttat caatcgaatc aatggattgt atgacaatgg aaattggaac
3240 ggttctattc gttcagatat ttcgtatcag aatatagacg cgattgtatt
accaacgtta 3300 ccaaagttac gccattggtt tatgtcagat agatttagtg
aacaaggaga tatcatggct 3360 aaattccaag gtgcattaaa tcgtgcgtat
gcacaactgg aacaaaatac gcttctgcat 3420 aatggtcatt ttacaaaaga
tgcagccaat tggacggtag aaggcgatgc acatcaggta 3480 gtattagaag
atggtaaacg tgtattacga ttgccagatt ggtcttcgag tgtgtctcaa 3540
acgattgaaa tcgagaattt tgatccagat aaagaatatc aattagtatt tcatgggcaa
3600 ggagaaggaa cggttacgtt ggagcatgga gaagaaacaa aatatataga
aacgcataca 3660 catcattttg cgaattttac aacttctcaa cgtcaaggac
tcacgtttga atcaaataaa 3720 gtgacagtga ccatttcttc agaagatgga
gaattcttag tggataatat tgcgcttgtg 3780 gaagctcctc ttcctacaga
tgaccaaaat tctgagggaa atacggcttc cagtacgaat 3840 agcgatacaa
gtatgaacaa caatcaa 3867 4 1289 PRT Bacillus thuringiensis 4 Met Ala
Ile Leu Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val Leu 1 5 10 15
Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp Ala Gly Thr Gln Ala Thr 20
25 30 Pro Ala Asp Leu Thr Ala Tyr Glu Gln Leu Leu Lys Asn Leu Glu
Lys 35 40 45 Gly Ile Asn Ala Gly Thr Tyr Ser Lys Ala Ile Ala Asp
Val Leu Lys 50 55 60 Gly Ile Phe Ile Asp Asp Thr Ile Asn Tyr Gln
Thr Tyr Val Asn Ile 65 70 75 80 Gly Leu Ser Leu Ile Thr Leu Ala Val
Pro Glu Ile Gly Ile Phe Thr 85 90 95 Pro Phe Ile Gly Leu Phe Phe
Ala Ala Leu Asn Lys His Asp Ala Pro 100 105 110 Pro Pro Pro Asn Ala
Lys Asp Ile Phe Glu Ala Met Lys Pro Ala Ile 115 120 125 Gln Glu Met
Ile Asp Arg Thr Leu Thr Ala Asp Glu Gln Thr Phe Leu 130 135 140 Asn
Gly Glu Ile Ser Gly Leu Gln Asn Leu Ala Ala Arg Tyr Gln Ser 145 150
155 160 Thr Met Asp Asp Ile Gln Ser His Gly Gly Phe Asn Lys Val Asp
Ser 165 170 175 Gly Leu Ile Lys Lys Phe Thr Asp Glu Val Leu Ser Leu
Asn Ser Phe 180 185 190 Tyr Thr Asp Arg Leu Pro Val Phe Ile Thr Asp
Asn Thr Ala Asp Arg 195 200 205 Thr Leu Leu Gly Leu Pro Tyr Tyr Ala
Ile Leu Ala Ser Met His Leu 210 215 220 Met Leu Leu Arg Asp Ile Ile
Thr Lys Gly Pro Thr Trp Asp Ser Lys 225 230 235 240 Ile Asn Phe Thr
Pro Asp Ala Ile Asp Ser Phe Lys Thr Asp Ile Lys 245 250 255 Asn Asn
Ile Lys Leu Tyr Ser Lys Thr Ile Tyr Asp Val Phe Gln Lys 260 265 270
Gly Leu Ala Ser Tyr Gly Thr Pro Ser Asp Leu Glu Ser Phe Ala Lys 275
280 285 Lys Gln Lys Tyr Ile Glu Ile Met Thr Thr His Cys Leu Asp Phe
Ala 290 295 300 Arg Leu Phe Pro Thr Phe Asp Pro Asp Leu Tyr Pro Thr
Gly Ser Gly 305 310 315 320 Asp Ile Ser Leu Gln Lys Thr Arg Arg Ile
Leu Ser Pro Phe Ile Pro 325 330 335 Ile Arg Thr Ala Asp Gly Leu Thr
Leu Asn Asn Thr Ser Ile Asp Thr 340 345 350 Ser Asn Trp Pro Asn Tyr
Glu Asn Gly Asn Gly Ala Phe Pro Asn Pro 355 360 365 Lys Glu Arg Ile
Leu Lys Gln Phe Lys Leu Tyr Pro Ser Trp Arg Ala 370 375 380 Ala Gln
Tyr Gly Gly Leu Leu Gln Pro Tyr Leu Trp Ala Ile Glu Val 385 390 395
400 Gln Asp Ser Val Glu Thr Arg Leu Tyr Gly Gln Leu Pro Ala Val Asp
405 410 415 Pro Gln Ala Gly Pro Asn Tyr Val Ser Ile Asp Ser Ser Asn
Pro Ile 420 425 430 Ile Gln Ile Asn Met Asp Thr Trp Lys Thr Pro Pro
Gln Gly Ala Ser 435 440 445 Gly Trp Asn Thr Asn Leu Met Arg Gly Ser
Val Ser Gly Leu Ser Phe 450 455 460 Leu Gln Arg Asp Gly Thr Arg Leu
Ser Ala Gly Met Gly Gly Gly Phe 465 470 475 480 Ala Asp Thr Ile Tyr
Ser Leu Pro Ala Thr His Tyr Leu Ser Tyr Leu 485 490 495 Tyr Gly Thr
Pro Tyr Gln Thr Ser Asp Asn Tyr Ser Gly His Val Gly 500 505 510 Ala
Leu Val Gly Val Ser Thr Pro Gln Glu Ala Thr Leu Pro Asn Ile 515 520
525 Ile Gly Gln Pro Asp Glu Gln Gly Asn Val Ser Thr Met Gly Phe Pro
530 535 540 Phe Glu Lys Ala Ser Tyr Gly Gly Thr Val Val Lys Glu Trp
Leu Asn 545 550 555 560 Gly Ala Asn Ala Met Lys Leu Ser Pro Gly Gln
Ser Ile Gly Ile Pro 565 570 575 Ile Thr Asn Val Thr Ser Gly Glu Tyr
Gln Ile Arg Cys Arg Tyr Ala 580 585 590 Ser Asn Asp Asn Thr Asn Val
Phe Phe Asn Val Asp Thr Gly Gly Ala 595 600 605 Asn Pro Ile Phe Gln
Gln Ile Asn Phe Ala Ser Thr Val Asp Asn Asn 610 615 620 Thr Gly Val
Gln Gly Ala Asn Gly Val Tyr Val Val Lys Ser Ile Ala 625 630 635 640
Thr Thr Asp Asn Ser Phe Thr Val Lys Ile Pro Ala Lys Thr Ile Asn 645
650 655 Val His Leu Thr Asn Gln Gly Ser Ser Asp Val Phe Leu Asp Arg
Ile 660 665 670 Glu Phe Val Pro Ile Leu Glu Ser Asn Thr Val Thr Ile
Phe Asn Asn 675 680 685 Ser Tyr Thr Thr Gly Ser Ala Asn Leu Ile Pro
Ala Ile Ala Pro Leu 690 695 700 Trp Ser Thr Ser Ser Asp Lys Ala Leu
Thr Gly Ser Met Ser Ile Thr 705 710 715 720 Gly Arg Thr Thr Pro Asn
Ser Asp Asp Ala Leu Leu Arg Phe Phe Lys 725 730 735 Thr Asn Tyr Asp
Thr Gln Thr Ile Pro Ile Pro Gly Ser Gly Lys Asp 740 745 750 Phe Thr
Asn Thr Leu Glu Ile Gln Asp Ile Val Ser Ile Asp Ile Phe 755 760 765
Val Gly Ser Gly Leu His Gly Ser Asp Gly Ser Ile Lys Leu Asp Phe 770
775 780 Thr Asn Asn Asn Ser Gly Ser Gly Gly Ser Pro Lys Ser Phe Thr
Glu 785 790 795 800 Gln Asn Asp Leu Glu Asn Ile Thr Thr Gln Val Asn
Ala Leu Phe Thr 805 810 815 Ser Asn Thr Gln Asp Ala Leu Ala Thr Asp
Val Ser Asp His Asp Ile 820 825 830 Glu Glu Val Val Leu Lys Val Asp
Ala Leu Ser Asp Glu Val Phe Gly 835 840 845 Lys Glu Lys Lys Thr Leu
Arg Lys Phe Val Asn Gln Ala Lys Arg Leu 850 855 860 Ser Lys Ala Arg
Asn Leu Leu Val Gly Gly Asn Phe Asp Asn Leu Asp 865 870 875 880 Ala
Trp Tyr Arg Gly Arg Asn Val Val Asn Val Ser Asn His Glu Leu 885 890
895 Leu Lys Ser Asp His Val Leu Leu Pro Pro Pro Gly Leu Ser Pro Ser
900 905 910 Tyr Ile Phe Gln Lys Val Glu Glu Ser Lys Leu Lys Arg Asn
Thr Arg 915 920 925 Tyr Thr Val Ser Gly Phe Ile Ala His Ala Thr Asp
Leu Glu Ile Val 930 935 940 Val Ser Arg Tyr Gly Gln Glu Ile Lys Lys
Val Val Gln Val Pro Tyr 945 950 955 960 Gly Glu Ala Phe Pro Leu Thr
Ser Ser Gly Pro Val Cys Cys Ile Pro 965 970 975 His Ser Thr Ser Asn
Gly Thr Leu Gly Asn Pro His Phe Phe Ser Tyr 980 985 990 Ser Ile Asp
Val Gly Ala Leu Asp Val Asp Thr Asn Pro Gly Ile Glu 995 1000 1005
Phe Gly Leu Arg Ile Val Asn Pro Thr Gly Met Ala Arg Val Ser 1010
1015 1020 Asn Leu Glu Ile Arg Glu Asp Arg Pro Leu Ala Ala Asn Glu
Ile 1025 1030 1035 Arg Gln Val Gln Arg Val Ala Arg Asn Trp Arg Thr
Glu Tyr Glu 1040 1045 1050 Lys Glu Arg Ala Glu Val Thr Ser Leu Ile
Gln Pro Val Ile Asn 1055 1060 1065 Arg Ile Asn Gly Leu Tyr Asp Asn
Gly Asn Trp Asn Gly Ser Ile 1070 1075 1080 Arg Ser Asp Ile Ser Tyr
Gln Asn Ile Asp Ala Ile Val Leu Pro 1085 1090 1095 Thr Leu Pro Lys
Leu Arg His Trp Phe Met Ser Asp Arg Phe Ser 1100 1105 1110 Glu Gln
Gly Asp Ile Met Ala Lys Phe Gln Gly Ala Leu Asn Arg 1115 1120 1125
Ala Tyr Ala Gln Leu Glu Gln Asn Thr Leu Leu His Asn Gly His 1130
1135 1140 Phe Thr Lys Asp Ala Ala Asn Trp Thr Val Glu Gly Asp Ala
His 1145 1150 1155 Gln Val Val Leu Glu Asp Gly Lys Arg Val Leu Arg
Leu Pro Asp 1160 1165 1170 Trp Ser Ser Ser Val Ser Gln Thr Ile Glu
Ile Glu Asn Phe Asp 1175 1180 1185 Pro Asp Lys Glu Tyr Gln Leu Val
Phe His Gly Gln Gly Glu Gly 1190 1195 1200 Thr Val Thr Leu Glu His
Gly Glu Glu Thr Lys Tyr Ile Glu Thr 1205 1210 1215 His Thr His His
Phe Ala Asn Phe Thr Thr Ser Gln Arg Gln Gly 1220 1225 1230 Leu Thr
Phe Glu Ser Asn Lys Val Thr Val Thr Ile Ser Ser Glu 1235 1240 1245
Asp Gly Glu Phe Leu Val Asp Asn Ile Ala Leu Val Glu Ala Pro 1250
1255 1260 Leu Pro Thr Asp Asp Gln Asn Ser Glu Gly Asn Thr Ala Ser
Ser 1265 1270 1275 Thr Asn Ser Asp Thr Ser Met Asn Asn Asn Gln 1280
1285 5 3771 DNA Bacillus thuringiensis misc_feature (4)..(24)
/function= "oligonucleotide hybridization probe" /product= "GCA/T
ACA/T TTA AAT GAA GTA/T TAT" /standard_name= "probe a" /note=
"probe a" 5 atggctacac ttaatgaagt atatcctgtg aattataatg tattatcttc
tgatgctttt 60 caacaattag atacaacagg ttttaaaagt aaatatgatg
aaatgataaa agcattcgaa 120 aaaaaatgga aaaaaggggc aaaaggaaaa
gaccttttag atgttgcatg gacttatata 180 actacaggag aaattgaccc
tttaaatgta attaaaggtg ttttatctgt attaacttta 240 attcctgaag
ttggtactgt ggcctctgca gcaagtacta ttgtaagttt tatttggcct 300
aaaatatttg gagataaacc aaatgcaaaa aatatatttg aagagctcaa gcctcaaatt
360 gaagcattaa ttcaacaaga tataacaaac tatcaagatg caattaatca
aaaaaaattt 420 gacagtcttc agaaaacaat taatctatat acagtagcta
tagataacaa tgattacgta 480 acagcaaaaa cgcaactcga aaatctaaat
tctatactta cctcagatat ctccatattt 540 attccagaag gatatgaaac
tggaggttta ccttattatg ctatggttgc taatgctcat 600 atattattgt
taagagacgc tatagttaat gcagagaaat taggctttag tgataaagaa 660
gtagacacac ataaaaaata tatcaaaatg acaatacaca atcatactga agcagtaata
720 aaagcattct taaatggact tgacaaattt aagagtttag atgtaaatag
ctataataaa 780 aaagcaaatt atattaaagg tatgacagaa atggttcttg
atctagttgc tctatggcca 840 actttcgatc cagatcatta tcaaaaagaa
gtagaaattg aatttacaag aactatttct 900 tctccaattt accaacctgt
acctaaaaac atgcaaaata cctctagctc tattgtacct 960 agcgatctat
ttcactatca aggagatctt gtaaaattag aattttctac aagaacggac 1020
aacgatggtc ttgcaaaaat ttttactggt attcgaaaca cattctacaa atcgcctaat
1080 actcatgaaa cataccatgt agattttagt tataataccc aatctagtgg
taatatttca 1140 agaggctctt caaatccgat tccaattgat cttaataatc
ccattatttc aacttgtatt 1200 agaaattcat tttataaggc aatagcggga
tcttctgttt tagttaattt taaagatggc 1260 actcaagggt atgcatttgc
ccaagcacca acaggaggtg cctgggacca ttcttttatt 1320 gaatctgatg
gtgccccaga agggcataaa ttaaactata tttatacttc tccaggtgat 1380
acattaagag atttcatcaa tgtatatact cttataagta ctccaactat aaatgaacta
1440 tcaacagaaa aaatcaaagg ctttcctgcg gaaaaaggat atatcaaaaa
tcaagggatc 1500 atgaaatatt acggtaaacc agaatatatt aatggagctc
aaccagttaa tctggaaaac 1560 cagcaaacat taatattcga atttcatgct
tcaaaaacag ctcaatatac cattcgtata 1620 cgttatgcca gtacccaagg
aacaaaaggt tattttcgtt tagataatca ggaactgcaa 1680 acgcttaata
tacctacttc acacaacggt tatgtaaccg gtaatattgg tgaaaattat 1740
gatttatata caataggttc atatacaatt acagaaggta accatactct tcaaatccaa
1800 cataatgata aaaatggaat ggttttagat cgtattgaat ttgttcctaa
agattcactt 1860 caagattcac ctcaagattc acctccagaa gttcacgaat
caacaattat ttttgataaa 1920 tcatctccaa ctatatggtc ttctaacaaa
cactcatata gccatataca tttagaagga 1980 tcatatacaa gtcagggaag
ttatccacac aatttattaa ttaatttatt tcatcctaca 2040 gaccctaaca
gaaatcatac tattcatgtt aacaatggtg atatgaatgt tgattatgga 2100
aaagattctg tagccgatgg gttaaatttt aataaaataa ctgctacgat accaagtgat
2160 gcttggtata gcggtactat tacttctatg cacttattta atgataataa
ttttaaaaca 2220 ataactccta aatttgaact ttctaatgaa ttagaaaaca
tcacaactca agtaaatgct 2280 ttattcgcat ctagtgcaca agatactctc
gcaagtaatg taagtgatta ctggattgaa 2340 caggtcgtta tgaaagtcga
tgccttatca gatgaagtat ttggaaaaga gaaaaaagca 2400 ttacgtaaat
tggtaaatca agcaaaacgt ctcagtaaaa tacgaaatct tctcataggt 2460
ggtaattttg acaatttagt cgcttggtat atgggaaaag atgtagtaaa agaatcggat
2520 catgaattat ttaaaagtga tcatgtctta ctacctcccc caacattcca
tccttcttat 2580 attttccaaa aggtggaaga atcaaaacta aaaccaaata
cacgttatac tatttctggt 2640 tttatcgcac atggagaaga tgtagagctt
gttgtctctc gttatgggca agaaatacaa 2700 aaagtgatgc aagtgccata
tgaagaagca cttcctctta catctgaatc taattctagt 2760 tgttgtgttc
caaatttaaa tataaatgaa acactagctg atccacattt ctttagttat 2820
agcatcgatg ttggttctct ggaaatggaa gcgaatcctg gtattgaatt tggtctccgt
2880 attgtcaaac caacaggtat ggcacgtgta agtaatttag aaattcgaga
agaccgtcca 2940 ttaacagcaa aagaaattcg tcaagtacaa cgtgcagcaa
gagattggaa acaaaactat 3000 gaacaagaac gaacagagat cacagctata
attcaacctg ttcttaatca aattaatgcg 3060 ttatacgaaa atgaagattg
gaatggttct attcgttcaa atgtttccta tcatgatcta 3120 gagcaaatta
tgcttcctac tttattaaaa actgaggaaa taaattgtaa ttatgatcat 3180
ccagcttttt tattaaaagt atatcattgg tttatgacag atcgtatagg agaacatggt
3240 actattttag cacgtttcca agaagcatta gatcgtgcat atacacaatt
agaaagtcgt 3300 aatctcctgc ataacggtca ttttacaact gatacagcga
attggacaat agaaggagat 3360 gcccatcata caatcttaga agatggtaga
cgtgtgttac gtttaccaga ttggtcttct 3420 aatgcaactc aaacaattga
aattgaagat tttgacttag atcaagaata ccaattgctc 3480 attcatgcaa
aaggaaaagg ttccattact ttacaacatg gagaagaaaa cgaatatgtg 3540
gaaacacata ctcatcatac aaatgatttt ataacatccc aaaatattcc tttcactttt
3600 aaaggaaatc aaattgaagt ccatattact tcagaagatg gagagttttt
aatcgatcac 3660 attacagtaa tagaagtttc taaaacagac acaaatacaa
atattattga aaattcacca 3720 atcaatacaa gtatgaatag taatgtaaga
gtagatatac caagaagtct c 3771 6 1257 PRT Bacillus thuringiensis 6
Met Ala Thr Leu Asn Glu Val Tyr Pro Val Asn Tyr Asn Val Leu Ser 1 5
10 15 Ser Asp Ala Phe Gln Gln Leu Asp Thr Thr Gly Phe Lys Ser Lys
Tyr 20 25 30 Asp Glu Met Ile Lys Ala Phe Glu Lys Lys Trp Lys Lys
Gly Ala Lys 35 40 45 Gly Lys Asp Leu Leu Asp Val Ala Trp Thr Tyr
Ile Thr Thr Gly Glu 50 55 60 Ile Asp Pro Leu Asn Val Ile Lys Gly
Val Leu Ser Val Leu Thr Leu 65 70 75 80 Ile Pro Glu Val Gly Thr Val
Ala Ser Ala Ala Ser Thr Ile Val Ser 85 90 95 Phe Ile Trp Pro Lys
Ile Phe Gly Asp Lys Pro Asn Ala Lys Asn Ile 100 105 110 Phe Glu Glu
Leu Lys Pro Gln Ile Glu Ala Leu Ile Gln Gln Asp Ile 115 120 125 Thr
Asn Tyr Gln Asp Ala Ile Asn Gln Lys Lys Phe Asp Ser Leu Gln 130 135
140 Lys Thr Ile Asn Leu Tyr Thr Val Ala Ile Asp Asn Asn Asp Tyr Val
145 150 155 160 Thr Ala Lys Thr Gln Leu Glu Asn Leu Asn Ser Ile Leu
Thr Ser Asp 165 170 175 Ile Ser Ile Phe Ile Pro Glu Gly Tyr Glu Thr
Gly Gly Leu Pro Tyr 180 185 190 Tyr Ala Met Val Ala Asn Ala His Ile
Leu Leu Leu Arg Asp Ala Ile 195 200 205 Val Asn Ala Glu Lys Leu Gly
Phe Ser Asp Lys Glu Val Asp Thr His 210 215 220 Lys Lys Tyr Ile
Lys Met Thr Ile His Asn His Thr Glu Ala Val Ile 225 230 235 240 Lys
Ala Phe Leu Asn Gly Leu Asp Lys Phe Lys Ser Leu Asp Val Asn 245 250
255 Ser Tyr Asn Lys Lys Ala Asn Tyr Ile Lys Gly Met Thr Glu Met Val
260 265 270 Leu Asp Leu Val Ala Leu Trp Pro Thr Phe Asp Pro Asp His
Tyr Gln 275 280 285 Lys Glu Val Glu Ile Glu Phe Thr Arg Thr Ile Ser
Ser Pro Ile Tyr 290 295 300 Gln Pro Val Pro Lys Asn Met Gln Asn Thr
Ser Ser Ser Ile Val Pro 305 310 315 320 Ser Asp Leu Phe His Tyr Gln
Gly Asp Leu Val Lys Leu Glu Phe Ser 325 330 335 Thr Arg Thr Asp Asn
Asp Gly Leu Ala Lys Ile Phe Thr Gly Ile Arg 340 345 350 Asn Thr Phe
Tyr Lys Ser Pro Asn Thr His Glu Thr Tyr His Val Asp 355 360 365 Phe
Ser Tyr Asn Thr Gln Ser Ser Gly Asn Ile Ser Arg Gly Ser Ser 370 375
380 Asn Pro Ile Pro Ile Asp Leu Asn Asn Pro Ile Ile Ser Thr Cys Ile
385 390 395 400 Arg Asn Ser Phe Tyr Lys Ala Ile Ala Gly Ser Ser Val
Leu Val Asn 405 410 415 Phe Lys Asp Gly Thr Gln Gly Tyr Ala Phe Ala
Gln Ala Pro Thr Gly 420 425 430 Gly Ala Trp Asp His Ser Phe Ile Glu
Ser Asp Gly Ala Pro Glu Gly 435 440 445 His Lys Leu Asn Tyr Ile Tyr
Thr Ser Pro Gly Asp Thr Leu Arg Asp 450 455 460 Phe Ile Asn Val Tyr
Thr Leu Ile Ser Thr Pro Thr Ile Asn Glu Leu 465 470 475 480 Ser Thr
Glu Lys Ile Lys Gly Phe Pro Ala Glu Lys Gly Tyr Ile Lys 485 490 495
Asn Gln Gly Ile Met Lys Tyr Tyr Gly Lys Pro Glu Tyr Ile Asn Gly 500
505 510 Ala Gln Pro Val Asn Leu Glu Asn Gln Gln Thr Leu Ile Phe Glu
Phe 515 520 525 His Ala Ser Lys Thr Ala Gln Tyr Thr Ile Arg Ile Arg
Tyr Ala Ser 530 535 540 Thr Gln Gly Thr Lys Gly Tyr Phe Arg Leu Asp
Asn Gln Glu Leu Gln 545 550 555 560 Thr Leu Asn Ile Pro Thr Ser His
Asn Gly Tyr Val Thr Gly Asn Ile 565 570 575 Gly Glu Asn Tyr Asp Leu
Tyr Thr Ile Gly Ser Tyr Thr Ile Thr Glu 580 585 590 Gly Asn His Thr
Leu Gln Ile Gln His Asn Asp Lys Asn Gly Met Val 595 600 605 Leu Asp
Arg Ile Glu Phe Val Pro Lys Asp Ser Leu Gln Asp Ser Pro 610 615 620
Gln Asp Ser Pro Pro Glu Val His Glu Ser Thr Ile Ile Phe Asp Lys 625
630 635 640 Ser Ser Pro Thr Ile Trp Ser Ser Asn Lys His Ser Tyr Ser
His Ile 645 650 655 His Leu Glu Gly Ser Tyr Thr Ser Gln Gly Ser Tyr
Pro His Asn Leu 660 665 670 Leu Ile Asn Leu Phe His Pro Thr Asp Pro
Asn Arg Asn His Thr Ile 675 680 685 His Val Asn Asn Gly Asp Met Asn
Val Asp Tyr Gly Lys Asp Ser Val 690 695 700 Ala Asp Gly Leu Asn Phe
Asn Lys Ile Thr Ala Thr Ile Pro Ser Asp 705 710 715 720 Ala Trp Tyr
Ser Gly Thr Ile Thr Ser Met His Leu Phe Asn Asp Asn 725 730 735 Asn
Phe Lys Thr Ile Thr Pro Lys Phe Glu Leu Ser Asn Glu Leu Glu 740 745
750 Asn Ile Thr Thr Gln Val Asn Ala Leu Phe Ala Ser Ser Ala Gln Asp
755 760 765 Thr Leu Ala Ser Asn Val Ser Asp Tyr Trp Ile Glu Gln Val
Val Met 770 775 780 Lys Val Asp Ala Leu Ser Asp Glu Val Phe Gly Lys
Glu Lys Lys Ala 785 790 795 800 Leu Arg Lys Leu Val Asn Gln Ala Lys
Arg Leu Ser Lys Ile Arg Asn 805 810 815 Leu Leu Ile Gly Gly Asn Phe
Asp Asn Leu Val Ala Trp Tyr Met Gly 820 825 830 Lys Asp Val Val Lys
Glu Ser Asp His Glu Leu Phe Lys Ser Asp His 835 840 845 Val Leu Leu
Pro Pro Pro Thr Phe His Pro Ser Tyr Ile Phe Gln Lys 850 855 860 Val
Glu Glu Ser Lys Leu Lys Pro Asn Thr Arg Tyr Thr Ile Ser Gly 865 870
875 880 Phe Ile Ala His Gly Glu Asp Val Glu Leu Val Val Ser Arg Tyr
Gly 885 890 895 Gln Glu Ile Gln Lys Val Met Gln Val Pro Tyr Glu Glu
Ala Leu Pro 900 905 910 Leu Thr Ser Glu Ser Asn Ser Ser Cys Cys Val
Pro Asn Leu Asn Ile 915 920 925 Asn Glu Thr Leu Ala Asp Pro His Phe
Phe Ser Tyr Ser Ile Asp Val 930 935 940 Gly Ser Leu Glu Met Glu Ala
Asn Pro Gly Ile Glu Phe Gly Leu Arg 945 950 955 960 Ile Val Lys Pro
Thr Gly Met Ala Arg Val Ser Asn Leu Glu Ile Arg 965 970 975 Glu Asp
Arg Pro Leu Thr Ala Lys Glu Ile Arg Gln Val Gln Arg Ala 980 985 990
Ala Arg Asp Trp Lys Gln Asn Tyr Glu Gln Glu Arg Thr Glu Ile Thr 995
1000 1005 Ala Ile Ile Gln Pro Val Leu Asn Gln Ile Asn Ala Leu Tyr
Glu 1010 1015 1020 Asn Glu Asp Trp Asn Gly Ser Ile Arg Ser Asn Val
Ser Tyr His 1025 1030 1035 Asp Leu Glu Gln Ile Met Leu Pro Thr Leu
Leu Lys Thr Glu Glu 1040 1045 1050 Ile Asn Cys Asn Tyr Asp His Pro
Ala Phe Leu Leu Lys Val Tyr 1055 1060 1065 His Trp Phe Met Thr Asp
Arg Ile Gly Glu His Gly Thr Ile Leu 1070 1075 1080 Ala Arg Phe Gln
Glu Ala Leu Asp Arg Ala Tyr Thr Gln Leu Glu 1085 1090 1095 Ser Arg
Asn Leu Leu His Asn Gly His Phe Thr Thr Asp Thr Ala 1100 1105 1110
Asn Trp Thr Ile Glu Gly Asp Ala His His Thr Ile Leu Glu Asp 1115
1120 1125 Gly Arg Arg Val Leu Arg Leu Pro Asp Trp Ser Ser Asn Ala
Thr 1130 1135 1140 Gln Thr Ile Glu Ile Glu Asp Phe Asp Leu Asp Gln
Glu Tyr Gln 1145 1150 1155 Leu Leu Ile His Ala Lys Gly Lys Gly Ser
Ile Thr Leu Gln His 1160 1165 1170 Gly Glu Glu Asn Glu Tyr Val Glu
Thr His Thr His His Thr Asn 1175 1180 1185 Asp Phe Ile Thr Ser Gln
Asn Ile Pro Phe Thr Phe Lys Gly Asn 1190 1195 1200 Gln Ile Glu Val
His Ile Thr Ser Glu Asp Gly Glu Phe Leu Ile 1205 1210 1215 Asp His
Ile Thr Val Ile Glu Val Ser Lys Thr Asp Thr Asn Thr 1220 1225 1230
Asn Ile Ile Glu Asn Ser Pro Ile Asn Thr Ser Met Asn Ser Asn 1235
1240 1245 Val Arg Val Asp Ile Pro Arg Ser Leu 1250 1255 7 1425 DNA
Artificial Sequence Clone E. coli NM522 (pMYC 2321) NRRL B-18770 7
atgattattg atagtaaaac gactttacct agacattcac ttattcatac aattaaatta
60 aattctaata agaaatatgg tcctggtgat atgactaatg gaaatcaatt
tattatttca 120 aaacaagaat gggctacgat tggagcatat attcagactg
gattaggttt accagtaaat 180 gaacaacaat taagaacaca tgttaattta
agtcaggata tatcaatacc tagtgatttt 240 tctcaattat atgatgttta
ttgttctgat aaaacttcag cagaatggtg gaataaaaat 300 ttatatcctt
taattattaa atctgctaat gatattgctt catatggttt taaagttgct 360
ggtgatcctt ctattaagaa agatggatat tttaaaaaat tgcaagatga attagataat
420 attgttgata ataattccga tgatgatgca atagctaaag ctattaaaga
ttttaaagcg 480 cgatgtggta ttttaattaa agaagctaaa caatatgaag
aagctgcaaa aaatattgta 540 acatctttag atcaattttt acatggtgat
cagaaaaaat tagaaggtgt tatcaatatt 600 caaaaacgtt taaaagaagt
tcaaacagct cttaatcaag cccatgggga aagtagtcca 660 gctcataaag
agttattaga aaaagtaaaa aatttaaaaa caacattaga aaggactatt 720
aaagctgaac aagatttaga gaaaaaagta gaatatagtt ttctattagg accattgtta
780 ggatttgttg tttatgaaat tcttgaaaat actgctgttc agcatataaa
aaatcaaatt 840 gatgagataa agaaacaatt agattctgct cagcatgatt
tggatagaga tgttaaaatt 900 ataggaatgt taaatagtat taatacagat
attgataatt tatatagtca aggacaagaa 960 gcaattaaag ttttccaaaa
gttacaaggt atttgggcta ctattggagc tcaaatagaa 1020 aatcttagaa
caacgtcgtt acaagaagtt caagattctg atgatgctga tgagatacaa 1080
attgaacttg aggacgcttc tgatgcttgg ttagttgtgg ctcaagaagc tcgtgatttt
1140 acactaaatg cttattcaac taatagtaga caaaatttac cgattaatgt
tatatcagat 1200 tcatgtaatt gttcaacaac aaatatgaca tcaaatcaat
acagtaatcc aacaacaaat 1260 atgacatcaa atcaatatat gatttcacat
gaatatacaa gtttaccaaa taattttatg 1320 ttatcaagaa atagtaattt
agaatataaa tgtcctgaaa ataattttat gatatattgg 1380 tataataatt
cggattggta taataattcg gattggtata ataat 1425 8 475 PRT Bacillus
thuringiensis 8 Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser
Leu Ile His 1 5 10 15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly
Pro Gly Asp Met Thr 20 25 30 Asn Gly Asn Gln Phe Ile Ile Ser Lys
Gln Glu Trp Ala Thr Ile Gly 35 40 45 Ala Tyr Ile Gln Thr Gly Leu
Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60 Arg Thr His Val Asn
Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 75 80 Ser Gln Leu
Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95 Trp
Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100 105
110 Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp
115 120 125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val
Asp Asn 130 135 140 Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys
Asp Phe Lys Ala 145 150 155 160 Arg Cys Gly Ile Leu Ile Lys Glu Ala
Lys Gln Tyr Glu Glu Ala Ala 165 170 175 Lys Asn Ile Val Thr Ser Leu
Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 Lys Leu Glu Gly Val
Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 Thr Ala Leu
Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 Leu
Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230
235 240 Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu
Leu 245 250 255 Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu
Asn Thr Ala 260 265 270 Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile
Lys Lys Gln Leu Asp 275 280 285 Ser Ala Gln His Asp Leu Asp Arg Asp
Val Lys Ile Ile Gly Met Leu 290 295 300 Asn Ser Ile Asn Thr Asp Ile
Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320 Ala Ile Lys Val
Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330 335 Ala Gln
Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345 350
Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355
360 365 Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn
Ala 370 375 380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val
Ile Ser Asp 385 390 395 400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr
Ser Asn Gln Tyr Ser Asn 405 410 415 Pro Thr Thr Asn Met Thr Ser Asn
Gln Tyr Met Ile Ser His Glu Tyr 420 425 430 Thr Ser Leu Pro Asn Asn
Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 Tyr Lys Cys Pro
Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 Asp Trp
Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 9 1185 DNA Bacillus
thuringiensis 9 atgattttag ggaatggaaa gactttacca aagcatataa
gattagctca tatttttgca 60 acacagaatt cttcagctaa gaaagacaat
cctcttggac cagaggggat ggttactaaa 120 gacggtttta taatctctaa
ggaagaatgg gcatttgtgc aggcctatgt gactacaggc 180 actggtttac
ctatcaatga cgatgagatg cgtagacatg ttgggttacc atcacgcatt 240
caaattcctg atgattttaa tcaattatat aaggtttata atgaagataa acatttatgc
300 agttggtgga atggtttctt gtttccatta gttcttaaaa cagctaatga
tatttccgct 360 tacggattta aatgtgctgg aaagggtgcc actaaaggat
attatgaggt catgcaagac 420 gatgtagaaa atatttcaga taatggttat
gataaagttg cacaagaaaa agcacataag 480 gatctgcagg cgcgttgtaa
aatccttatt aaggaggctg atcaatataa agctgcagcg 540 gatgatgttt
caaaacattt aaacacattt cttaaaggcg gtcaagattc agatggcaat 600
gatgttattg gcgtagaggc tgttcaagta caactagcac aagtaaaaga taatcttgat
660 ggcctatatg gcgacaaaag cccaagacat gaagagttac taaagaaagt
agacgacctg 720 aaaaaagagt tggaagctgc tattaaagca gagaatgaat
tagaaaagaa agtgaaaatg 780 agttttgctt taggaccatt acttggattt
gttgtatatg aaatcttaga gctaactgcg 840 gtcaaaagta tacacaagaa
agttgaggca ctacaagccg agcttgacac tgctaatgat 900 gaactcgaca
gagatgtaaa aatcttagga atgatgaata gcattgacac tgatattgac 960
aacatgttag agcaaggtga gcaagctctt gttgtattta gaaaaattgc aggcatttgg
1020 agtgttataa gtcttaatat cggcaatctt cgagaaacat ctttaaaaga
gatagaagaa 1080 gaaaatgatg acgatgcact gtatattgag cttggtgatg
ccgctggtca atggaaagag 1140 atagccgagg aggcacaatc ctttgtacta
aatgcttata ctcct 1185 10 395 PRT Bacillus thuringiensis 10 Met Ile
Leu Gly Asn Gly Lys Thr Leu Pro Lys His Ile Arg Leu Ala 1 5 10 15
His Ile Phe Ala Thr Gln Asn Ser Ser Ala Lys Lys Asp Asn Pro Leu 20
25 30 Gly Pro Glu Gly Met Val Thr Lys Asp Gly Phe Ile Ile Ser Lys
Glu 35 40 45 Glu Trp Ala Phe Val Gln Ala Tyr Val Thr Thr Gly Thr
Gly Leu Pro 50 55 60 Ile Asn Asp Asp Glu Met Arg Arg His Val Gly
Leu Pro Ser Arg Ile 65 70 75 80 Gln Ile Pro Asp Asp Phe Asn Gln Leu
Tyr Lys Val Tyr Asn Glu Asp 85 90 95 Lys His Leu Cys Ser Trp Trp
Asn Gly Phe Leu Phe Pro Leu Val Leu 100 105 110 Lys Thr Ala Asn Asp
Ile Ser Ala Tyr Gly Phe Lys Cys Ala Gly Lys 115 120 125 Gly Ala Thr
Lys Gly Tyr Tyr Glu Val Met Gln Asp Asp Val Glu Asn 130 135 140 Ile
Ser Asp Asn Gly Tyr Asp Lys Val Ala Gln Glu Lys Ala His Lys 145 150
155 160 Asp Leu Gln Ala Arg Cys Lys Ile Leu Ile Lys Glu Ala Asp Gln
Tyr 165 170 175 Lys Ala Ala Ala Asp Asp Val Ser Lys His Leu Asn Thr
Phe Leu Lys 180 185 190 Gly Gly Gln Asp Ser Asp Gly Asn Asp Val Ile
Gly Val Glu Ala Val 195 200 205 Gln Val Gln Leu Ala Gln Val Lys Asp
Asn Leu Asp Gly Leu Tyr Gly 210 215 220 Asp Lys Ser Pro Arg His Glu
Glu Leu Leu Lys Lys Val Asp Asp Leu 225 230 235 240 Lys Lys Glu Leu
Glu Ala Ala Ile Lys Ala Glu Asn Glu Leu Glu Lys 245 250 255 Lys Val
Lys Met Ser Phe Ala Leu Gly Pro Leu Leu Gly Phe Val Val 260 265 270
Tyr Glu Ile Leu Glu Leu Thr Ala Val Lys Ser Ile His Lys Lys Val 275
280 285 Glu Ala Leu Gln Ala Glu Leu Asp Thr Ala Asn Asp Glu Leu Asp
Arg 290 295 300 Asp Val Lys Ile Leu Gly Met Met Asn Ser Ile Asp Thr
Asp Ile Asp 305 310 315 320 Asn Met Leu Glu Gln Gly Glu Gln Ala Leu
Val Val Phe Arg Lys Ile 325 330 335 Ala Gly Ile Trp Ser Val Ile Ser
Leu Asn Ile Gly Asn Leu Arg Glu 340 345 350 Thr Ser Leu Lys Glu Ile
Glu Glu Glu Asn Asp Asp Asp Ala Leu Tyr 355 360 365 Ile Glu Leu Gly
Asp Ala Ala Gly Gln Trp Lys Glu Ile Ala Glu Glu 370 375 380 Ala Gln
Ser Phe Val Leu Asn Ala Tyr Thr Pro 385 390 395 11 2412 DNA
Bacillus thuringiensis 11 atgacttgtc aattacaagc gcaaccactt
attccctata acgtactagc aggagttcca 60 actagtaata caggtagtcc
aatcggcaat gcaggtaatc aatttgatca gtttgagcaa 120 accgttaaag
agctcaagga agcatgggaa gcgttccaaa aaaacggaag tttctcatta 180
gcagctcttg aaaagggatt tgatgcagca atcggaggag gatcctttga ttatttaggt
240 ttagttcaag ccggcctagg attagttggt acgctaggcg ccgcaatccc
tggtgtttca 300 gtggcagtgc ctcttattag catgcttgtt ggtgtttttt
ggccaaaggg cacaaacaac 360 caagaaaacc ttattacagt tattgataag
gaagttcaga gaatactaga tgaaaagcta 420 tctgatcagt taataaagaa
attgaacgca gatttaaatg cttttacgga cctagtaact 480 cgtttggaag
aagtaataat agatgcaact ttcgagaatc acaagcctgt
actacaagta 540 agtaaatcaa attatatgaa agtggattca gcatatttct
caacaggagg tattcttact 600 cttggcatga gtgattttct tactgatacc
tattcaaagc ttaccttccc attatatgta 660 ctaggcgcaa ctatgaaact
ttcagcatat catagttata tacaattcgg aaatacatgg 720 cttaataaag
tttatgattt atcatcagat gagggaaaaa caatgtcgca ggctttagca 780
cgagctaaac agcatatgcg ccaagacata gcattttata caagccaagc tttaaacatg
840 tttactggga atctcccttc attatcatct aataaatatg caattaatga
ctataatgta 900 tacactcgag caatggtatt gaatggctta gatatagtag
caacatggcc taccctatat 960 ccagatgact attcgtctca gataaaactg
gagaaaacac gcgtgatctt ttcagatatg 1020 gtcgggcaaa gtgagagtag
agatggcagc gtaacgatta aaaatatttt tgacaataca 1080 gattcacatc
aacatggatc cataggtctc aattcaatct cttatttccc agatgagtta 1140
cagaaagcac aacttcgcat gtatgattat aatcacaaac cttattgtac ggactgtttc
1200 tgctggccgt atggagtgat tttaaactat aacaagaata cctttagata
tggcgataat 1260 gatccaggtc tttcaggaga cgttcaactc ccagcaccta
tgagtgtagt taatgcccaa 1320 actcaaacag cccaatatac agatggagaa
aacatatgga cagatactgg ccgcagttgg 1380 ctttgtactc tacgtggcta
ctgtactaca aactgttttc caggaagagg ttgttataat 1440 aatagtactg
gatatggaga aagttgcaat caatcacttc caggtcaaaa aatacatgca 1500
ctatatcctt ttacacaaac aaatgtgctg ggacaatcag gcaaactagg attgctagca
1560 agtcatattc catatgacct aagtccgaac aatacgattg gtgacaaaga
tacagattct 1620 acgaatattg tcgcaaaagg aattccagtg gaaaaagggt
atgcatccag tggacaaaaa 1680 gttgaaatta tacgagagtg gataaatggt
gcgaatgtag ttcaattatc tccaggccaa 1740 tcttggggaa tggattttac
caatagcaca ggtggtcaat atatggtccg ctgtcgatat 1800 gcaagtacaa
acgatactcc aatctttttt aatttagtgt atgacggggg atcgaatcct 1860
atttataacc agatgacatt ccctgctaca aaagagactc cagctcacga ttcagtagat
1920 aacaagatac taggcataaa aggaataaat ggaaattatt cactcatgaa
tgtaaaagat 1980 tctgtcgaac ttccatctgg gaaatttcat gtttttttca
caaataatgg atcatctgct 2040 atttatttag atcgacttga gtttgttcct
ttagatcaac cagcagcgcc aacacagtca 2100 acacaaccaa ttaattatcc
tatcacaagt aggttacctc atcgttccgg agaaccacct 2160 gcaataatat
gggagaaatc agggaatgtt cgcgggaatc aactaactat atcggcacaa 2220
ggtgttccag aaaattccca aatatatctt tcggtgggtg gcgatcgcca aattttagac
2280 cgtagcaacg gatttaaatt agttaattac tcacctactt attctttcac
taacattcag 2340 gctagctcgt caaatttagt agatattaca agtggtacca
tcactggcca agtacaagta 2400 tctaatctat aa 2412 12 803 PRT Bacillus
thuringiensis 12 Met Thr Cys Gln Leu Gln Ala Gln Pro Leu Ile Pro
Tyr Asn Val Leu 1 5 10 15 Ala Gly Val Pro Thr Ser Asn Thr Gly Ser
Pro Ile Gly Asn Ala Gly 20 25 30 Asn Gln Phe Asp Gln Phe Glu Gln
Thr Val Lys Glu Leu Lys Glu Ala 35 40 45 Trp Glu Ala Phe Gln Lys
Asn Gly Ser Phe Ser Leu Ala Ala Leu Glu 50 55 60 Lys Gly Phe Asp
Ala Ala Ile Gly Gly Gly Ser Phe Asp Tyr Leu Gly 65 70 75 80 Leu Val
Gln Ala Gly Leu Gly Leu Val Gly Thr Leu Gly Ala Ala Ile 85 90 95
Pro Gly Val Ser Val Ala Val Pro Leu Ile Ser Met Leu Val Gly Val 100
105 110 Phe Trp Pro Lys Gly Thr Asn Asn Gln Glu Asn Leu Ile Thr Val
Ile 115 120 125 Asp Lys Glu Val Gln Arg Ile Leu Asp Glu Lys Leu Ser
Asp Gln Leu 130 135 140 Ile Lys Lys Leu Asn Ala Asp Leu Asn Ala Phe
Thr Asp Leu Val Thr 145 150 155 160 Arg Leu Glu Glu Val Ile Ile Asp
Ala Thr Phe Glu Asn His Lys Pro 165 170 175 Val Leu Gln Val Ser Lys
Ser Asn Tyr Met Lys Val Asp Ser Ala Tyr 180 185 190 Phe Ser Thr Gly
Gly Ile Leu Thr Leu Gly Met Ser Asp Phe Leu Thr 195 200 205 Asp Thr
Tyr Ser Lys Leu Thr Phe Pro Leu Tyr Val Leu Gly Ala Thr 210 215 220
Met Lys Leu Ser Ala Tyr His Ser Tyr Ile Gln Phe Gly Asn Thr Trp 225
230 235 240 Leu Asn Lys Val Tyr Asp Leu Ser Ser Asp Glu Gly Lys Thr
Met Ser 245 250 255 Gln Ala Leu Ala Arg Ala Lys Gln His Met Arg Gln
Asp Ile Ala Phe 260 265 270 Tyr Thr Ser Gln Ala Leu Asn Met Phe Thr
Gly Asn Leu Pro Ser Leu 275 280 285 Ser Ser Asn Lys Tyr Ala Ile Asn
Asp Tyr Asn Val Tyr Thr Arg Ala 290 295 300 Met Val Leu Asn Gly Leu
Asp Ile Val Ala Thr Trp Pro Thr Leu Tyr 305 310 315 320 Pro Asp Asp
Tyr Ser Ser Gln Ile Lys Leu Glu Lys Thr Arg Val Ile 325 330 335 Phe
Ser Asp Met Val Gly Gln Ser Glu Ser Arg Asp Gly Ser Val Thr 340 345
350 Ile Lys Asn Ile Phe Asp Asn Thr Asp Ser His Gln His Gly Ser Ile
355 360 365 Gly Leu Asn Ser Ile Ser Tyr Phe Pro Asp Glu Leu Gln Lys
Ala Gln 370 375 380 Leu Arg Met Tyr Asp Tyr Asn His Lys Pro Tyr Cys
Thr Asp Cys Phe 385 390 395 400 Cys Trp Pro Tyr Gly Val Ile Leu Asn
Tyr Asn Lys Asn Thr Phe Arg 405 410 415 Tyr Gly Asp Asn Asp Pro Gly
Leu Ser Gly Asp Val Gln Leu Pro Ala 420 425 430 Pro Met Ser Val Val
Asn Ala Gln Thr Gln Thr Ala Gln Tyr Thr Asp 435 440 445 Gly Glu Asn
Ile Trp Thr Asp Thr Gly Arg Ser Trp Leu Cys Thr Leu 450 455 460 Arg
Gly Tyr Cys Thr Thr Asn Cys Phe Pro Gly Arg Gly Cys Tyr Asn 465 470
475 480 Asn Ser Thr Gly Tyr Gly Glu Ser Cys Asn Gln Ser Leu Pro Gly
Gln 485 490 495 Lys Ile His Ala Leu Tyr Pro Phe Thr Gln Thr Asn Val
Leu Gly Gln 500 505 510 Ser Gly Lys Leu Gly Leu Leu Ala Ser His Ile
Pro Tyr Asp Leu Ser 515 520 525 Pro Asn Asn Thr Ile Gly Asp Lys Asp
Thr Asp Ser Thr Asn Ile Val 530 535 540 Ala Lys Gly Ile Pro Val Glu
Lys Gly Tyr Ala Ser Ser Gly Gln Lys 545 550 555 560 Val Glu Ile Ile
Arg Glu Trp Ile Asn Gly Ala Asn Val Val Gln Leu 565 570 575 Ser Pro
Gly Gln Ser Trp Gly Met Asp Phe Thr Asn Ser Thr Gly Gly 580 585 590
Gln Tyr Met Val Arg Cys Arg Tyr Ala Ser Thr Asn Asp Thr Pro Ile 595
600 605 Phe Phe Asn Leu Val Tyr Asp Gly Gly Ser Asn Pro Ile Tyr Asn
Gln 610 615 620 Met Thr Phe Pro Ala Thr Lys Glu Thr Pro Ala His Asp
Ser Val Asp 625 630 635 640 Asn Lys Ile Leu Gly Ile Lys Gly Ile Asn
Gly Asn Tyr Ser Leu Met 645 650 655 Asn Val Lys Asp Ser Val Glu Leu
Pro Ser Gly Lys Phe His Val Phe 660 665 670 Phe Thr Asn Asn Gly Ser
Ser Ala Ile Tyr Leu Asp Arg Leu Glu Phe 675 680 685 Val Pro Leu Asp
Gln Pro Ala Ala Pro Thr Gln Ser Thr Gln Pro Ile 690 695 700 Asn Tyr
Pro Ile Thr Ser Arg Leu Pro His Arg Ser Gly Glu Pro Pro 705 710 715
720 Ala Ile Ile Trp Glu Lys Ser Gly Asn Val Arg Gly Asn Gln Leu Thr
725 730 735 Ile Ser Ala Gln Gly Val Pro Glu Asn Ser Gln Ile Tyr Leu
Ser Val 740 745 750 Gly Gly Asp Arg Gln Ile Leu Asp Arg Ser Asn Gly
Phe Lys Leu Val 755 760 765 Asn Tyr Ser Pro Thr Tyr Ser Phe Thr Asn
Ile Gln Ala Ser Ser Ser 770 775 780 Asn Leu Val Asp Ile Thr Ser Gly
Thr Ile Thr Gly Gln Val Gln Val 785 790 795 800 Ser Asn Leu 13 8
PRT Artificial sequence Probe 13 Arg Glu Trp Ile Asn Gly Ala Asn 1
5 14 22 DNA Artificial sequence DNA coding for probe of SEQ ID NO13
14 agartrkwtw aatggwgckm aw 22 15 8 PRT Artificial Sequence Probe
15 Pro Thr Phe Asp Pro Asp Leu Tyr 1 5 16 24 DNA Artificial
Sequence DNA coding for probe of SEQ ID NO15 16 ccnacytttk
atccagatsw ytat 24 17 14 PRT Artificial Sequence N-terminal amino
acid sequence of 17a. 17 Ala Ile Leu Asn Glu Leu Tyr Pro Ser Val
Pro Tyr Asn Val 1 5 10 18 14 PRT Artificial Sequence N-terminal
amino acid sequence of 17b. 18 Ala Ile Leu Asn Glu Leu Tyr Pro Ser
Val Pro Tyr Asn Val 1 5 10 19 17 PRT Artificial Sequence N-terminal
amino acid sequence of 52A1. 19 Met Ile Ile Asp Ser Lys Thr Thr Leu
Pro Arg His Ser Leu Ile Asn 1 5 10 15 Thr 20 14 PRT Artificial
Sequence N-terminal amino acid sequence of 63B. 20 Gln Leu Gln Ala
Gln Pro Leu Ile Pro Tyr Asn Val Leu Ala 1 5 10 21 24 PRT Artificial
Sequence N-terminal amino acid sequence of 69D1. 21 Met Ile Leu Gly
Asn Gly Lys Thr Leu Pro Lys His Ile Arg Leu Ala 1 5 10 15 His Ile
Phe Ala Thr Gln Asn Ser 20 22 10 PRT Artificial Sequence N-terminal
amino acid sequence of 33F2. 22 Ala Thr Leu Asn Glu Val Tyr Pro Val
Asn 1 5 10 23 15 PRT Artificial Sequence Internal amino acid
sequence for 63B. 23 Val Gln Arg Ile Leu Asp Glu Lys Leu Ser Phe
Gln Leu Ile Lys 1 5 10 15 24 23 DNA Artificial Sequence Synthetic
oligonucleotide derived from 17. 24 gcaattttaa atgaattata tcc 23 25
56 DNA Artificial Sequence Oligonucleotide probe designed from the
N-terminal amino acid sequence of 52A1. 25 atgattattg attctaaaac
aacattacca agacattcwt taatwaatac watwaa 56 26 38 DNA Artificial
Sequence Synthetic oligonucleotide probe designated as 69D1-D. 26
aaacatatta gattagcaca tatttttgca acacaaaa 38 27 17 DNA Artificial
Sequence Forward oligonucleotide primer from 63B. 27 caaytacaag
cwcaacc 17 28 21 DNA Artificial Sequence Reverse oligonucleotide
primer from 63B. 28 ttcatctaaa attctttgwa c 21 29 8 PRT Artificial
Sequence Nematode (NEMI) variant of region 5 of Hofte and Whiteley.
29 Leu Asp Arg Ile Gln Phe Ile Pro 1 5 30 23 DNA Artificial
Sequence Reverse complement primer to SEQ ID NO29. 30 aggaacaaay
tcaakwcgrt cta 23 31 9 PRT Artificial Sequence Peptide 31 Tyr Ile
Asp Lys Ile Glu Phe Ile Pro 1 5 32 23 DNA Artificial Sequence
Oligonucleotide coding for the peptide of SEQ ID NO31. 32
tggaataaat tcaattykrt cwa 23 33 21 DNA Artificial Sequence
Oligonucleotide probe 33F2A. 33 gcwacwttaa atgaagtwta t 21 34 21
DNA Artificial Sequence Oligonucleotide probe 33F2B. 34 aatgaagtwt
atccwgtwaa t 21 35 38 DNA Artificial sequence Reverse primer. 35
gcaagcggcc gcttatggaa taaattcaat tykrtcwa 38 36 28 DNA Artificial
Sequence Forward primer. 36 tgattttwmt caattatatr akgtttat 28 37 20
DNA Artificial Sequence Probe 37 aagagttayt araraaagta 20 38 35 DNA
Artificial Sequence Probe 38 ttaggaccat trytwggatt tgttgtwtat gaaat
35 39 27 DNA Artificial Sequence Probe 39 gayagagatg twaaaatywt
aggaatg 27 40 23 DNA Artificial Sequence Forward primer. 40
ttmttaaawc wgctaatgat att 23
* * * * *